Manganese ion regulation of reverse transcriptase activity and methods of modulating same

ABSTRACT

Methods of identifying agents that modulate reverse transcriptase activity in a cell by affecting manganese ion transport across a membrane of the cell are provided, as are agents identified using such methods. Also provided are methods of modulating reverse transcriptase activity by affecting manganese ion concentration. In addition, methods of reducing or inhibiting infection of cells with a retrotransposable element are provided.

This application claims the benefit of priority under 35 U.S.C. §365 ofPCT/US03/07879 filed Mar. 12, 2003 and under 35 U.S.C. § 119(e)(1) ofU.S. Ser. No. 60/363,708, filed Mar. 12, 2002, the entire content ofwhich is incorporated herein by reference.

This invention was made in part with government support under Grant No.GM 36481 awarded by the National Institutes of Health. The United Statesgovernment has certain rights in this invention.

BACKGROUND OF THE INVENTION

1. Field of the Invention

The invention relates generally to methods of manipulating manganeselevels to alter reverse transcriptase activity, and more specifically tomethods of identifying agents that modulate reverse transcriptaseactivity, to agents identified using such methods, and to methods ofusing such agents to modulate reverse transcriptase activity, forexample, in a cell.

2. Background Information

Viral infection and replication in host cells is associated with variousdiseases in plants and animals. Retroviruses, for example, are a type ofvirus that associated with various cancer and with humanimmunodeficiency virus (HIV), which is responsible for AIDS in humans.As such, the morbidity and mortality associated with viral infectionsand the disease associated with such infections causes great sufferingand further results in a great economic burden on individuals andsociety.

HIV was identified as the causative agent of AIDS in 1983 and, AIDS hasprogressed to being one of the greatest health problems in the world,with medical and social consequences likely to extend long into thefuture. The World Health Organization has estimated that between eightand ten million people are currently infected with HIV, and thatapproximately ten times as many individuals will be affected in the nextdecade. Further, the large pool of HIV carriers and the failure of HIVinfection to cause early and easily identified symptoms makes thedevelopment of effective antiviral treatments a medical priority.

Retroviruses such as HIV replicate through an RNA intermediate as partof their life cycle. All retroviruses encode proteins that are requiredfor their replication and transmission, including, for example, anintegrase that allows the virus to integrate into the genomic DNA of aninfected cell, and a reverse transcriptase that is involved inreplication of the retrovirus genome. The activities of these retroviralproteins, including the reverse transcriptase, are central to thereplication of retroviruses and, therefore, have been the target ofdrugs to treat retroviral infection, including HIV infection. Reversetranscriptase inhibitors are the current treatment of choice for AIDSpatients. However, retroviral therapy, as for other viral therapies, arecommonly of limited effectiveness due to development of viral resistanceto the drug.

In an effort to overcome problems associated with the development ofresistance to a single drug, “cocktails” containing a combination ofdrugs have been used, and have been shown to be effective for longerperiods of time. Unfortunately, drugs that are effective in inhibitingthe activity of retroviral proteins often inhibit normal proteinsinvolved in survival and proliferation of the retroviral infected cells,thus causing undesirable and often severe side effects. Thus, a needexists for developing agents and methods for more effectively andspecifically inhibiting retroviral enzymes without substantiallyaffecting normal cellular enzymes. The present invention satisfies thisneed provides additional advantages.

SUMMARY OF THE INVENTION

The present invention relates to a method of identifying an agent thatmodulates reverse transcriptase activity in a cell. In one embodiment, amethod of the invention can be performed, for example, contacting a cellmembrane, which contains a divalent cation transporting protein thattransports manganese ions, and can transport other divalent cations,with a test agent; and detecting altered manganese ion transport due tocontact with the test agent as compared to manganese ion transport inthe absence of the test agent. The cell membrane contacted with the testagent can be an isolated cell membrane, which contains the divalentcation transporting protein, for example, a eukaryotic cell membranesuch as a yeast cell membrane or a mammalian cell membrane (e.g., ahuman cell membrane), and can be any cell membrane of a cell, including,for example, the cell surface membrane, or a cell membrane of associatedwith the Golgi apparatus, mitochondria, endoplasmic reticulum, ornucleus.

Generally, an isolated cell membrane useful in a method of the inventiondelimits at least a first compartment and a second compartment, whereinthe divalent cation transporting protein in the membrane can transportdivalent cations, including manganese, from the first compartment to thesecond, or from the second compartment to the first; or can transportdivalent cations into and out of both compartments, includingtransporting manganese into and/or out of at least one compartment. Forexample, a portion of a cell membrane can be obtained using amicrocapillary, wherein the cell membrane can separate the contents ofthe microcapillary (i.e., a first compartment) from a medium in whichthe microcapillary is contacted (i.e., a second compartment). Accordingto such an example, the test agent can present in the medium (secondcompartment), manganese ions can be present in the first and/or secondcompartment, and altered manganese ion transport can be detected byexamining the first compartment or the second compartment or both.

The cell membrane contacted with the test agent also can be a cellmembrane in situ, in which case the method is performed by contacting acell, which comprises the cell membrane. The cell can be any type ofcell that contains a cell membrane with a divalent cation transportingprotein and that supports reverse transcriptase activity. As such, thecell can be a eukaryotic cell, including, for example, an insect cell(e.g., a Drosophila cell), a fungus cell (e.g., a Neurospora cell), ayeast cell, a C. elegans cell, an amphibian cell (e.g., sea urchin), anavian cell (e.g., a chick embryo fibroblast), or a human cell (e.g., ahuman T lymphocyte). Further, such cells useful in a method of theinvention can be cells of a cell line, which have been adapted toculture; can be cells of a primary cell culture, which can be maintainedin culture for at least a short period of time; or cells that have beenisolated from a living organism, for example, cells isolated from ahuman subject.

The divalent cation transporting protein of the cell membrane can be anytransporter that allows the selective passage of manganese ions acrossthe cell membrane. The transporter generally is an active transporterthat normally functions to pump manganese ions into a cell, out of acell, or in either direction depending, for example, on the relativeconcentration of manganese ions in one of the compartments, particularlyan intracellular compartment, as compared to a manganese concentrationtypically found in the compartment in an otherwise normal cell. Forexample, the divalent cation transporting protein can be a P-typeATPase, for example, a Saccharomyces cerevisiae Pmr1p protein, which isa calcium ion and manganese ion transporting protein, or a homologthereof, including for example, isoforms of the human ATP-dependentcalcium ion pump, PMR1 (ATPase 2C1); the Pmr1p homologs ATP2 μl andATP2A2, which are expressed in cardiac cells; and ATP2A3, which isexpressed ubiquitously.

An agent that modulates reverse transcriptase activity, as identifiedaccording to a method of the invention, can be one that alters thetransport of all cations that are transported by the divalent cationtransporting protein, or, in particular, an agent that only altersmanganese ion transport, but not any other divalent cations (if any)that can be transported by the divalent cation transporting protein.Further, the method can be used to identify an agent that reduces orinhibits manganese ion transport out of a cell, or that increasesmanganese transport into a cell, thus providing an agent that canincrease an intracellular manganese ion concentration in a cell above alevel normally found in the cell; or the method can be used to identifyan agent that reduces or inhibits manganese ion transport into a cell,or that increase manganese ion transport out of a cell, thus providingan agent that can decrease an intracellular manganese ion concentrationin a cell below a level normally found in the cell.

Altered manganese ion transport can be detected using any of variousmethods useful for detecting the presence or absence, or theconcentration or relative amount of manganese ion, in a compartment,including in vivo (i.e., in a cell) in an intracellular compartment, orin vitro (i.e., using an isolated cell membrane) in a compartmentdelimited by the cell membrane. As such, manganese ion concentration inone (or more) compartments can be measured using a chemical or physicalmeans, for example, using a polarographic (voltametric) method or aradiometric method. In addition, or alternatively, as disclosed herein,altered manganese ion transport can be detected by detecting alteredreverse transcriptase activity in a relevant compartment, which can bein intracellular compartment or other compartment delimited by the cellmembrane. Reverse transcriptase activity can be measured using apolyribonucleotide template, or a polydeoxyribonucleotide template, orboth. Further, the reverse transcriptase activity being detected can bethat of a reverse transcriptase that is present in a cell due, forexample, to infection of the cell by a retrotransposon that expressesthe reverse transcriptase, or to expression in a cell of an exogenouslyadded nucleic acid molecule encoding the reverse transcriptase, or canbe that of an isolated reverse transcriptase polypeptide.

In another embodiment, a method of identifying an agent that modulatesreverse transcriptase activity in a cell can be performed, for example,by contacting a cell expressing a reverse transcriptase with a testagent; and detecting altered reverse transcriptase activity due tocontact with the test agent as compared to reverse transcriptaseactivity in the absence of the test agent, wherein the test agent altersmanganese ion concentration in the cell. A test agent useful in a methodof the invention can be any agent suspected of having the ability toalter manganese ion transport across a cell membrane, including anyagent suspected of having the ability to alter the activity of adivalent cation transporting protein. As such, the test agent can be apeptide, a polynucleotide, a small organic molecule, a peptidomimetic,or the like.

Altered reverse transcriptase activity due to contact with a test agentcomprises measuring cDNA produced by the reverse transcriptase using apolynucleotide template in the cell. The polynucleotide template can bea polydeoxyribonucleotide template or a polyribonucleotide template. Inone embodiment, the polynucleotide template comprises a nucleotidesequence of a retrotransposable element, for example, a nucleotidesequence of a retrotransposon such as a Ty retrotransposon (e.g., a Ty-1element); or a nucleotide sequence of a retrovirus such as a humanimmunodeficiency virus (HIV; e.g., HIV-1) or an avian myeloblastosisvirus. In another embodiment, the reverse transcriptase is a reversetranscriptase expressed in a cell from a retrotransposable elementpresent in the cell, for example, a Ty-1 element or a retrovirus such asHIV-1.

The cell contacted with the agent can be any cell as disclosed herein,for example, a yeast cell (e.g., a S. cerevisiae cell), an avian cell(e.g., a chick embryo fibroblast), or a human or other mammalian cell(e.g., a T lymphocyte), including cells isolated from a subject.Further, the cell can be one of a plurality of cells, which can be thesame or different or a combination of some that are the same and somethat are different, wherein, preferably, cells of the plurality aresubstantially isolated from each other. As such, the methods of theinvention can be adapted so as to be performed in a high throughputformat. In one embodiment, each of the cells of the plurality isarranged in an array, which can be an addressable array, for example, ona solid support such as a microchip, on a glass slide, on a bead, or ina well. In another embodiment, each of the cells of the plurality iscontacted with a test agent, which, in various aspects, can includecontacting two or more cells of the plurality that are the same with thesame test agent (thus providing duplicates, triplicates, etc., screenedin parallel) or with different test agents (thus providing a means toexamine a variety of different test agents, e.g., each test agent of alibrary of random, biased or variegated test agents); or can includecontacting two or more cells of the plurality that are different withthe same test agent (thus providing a means to determine the effect of atest agent on different cells or cell types) or with different testagents (thus providing a means to examine the effect of a variety ofdifferent test agents on a variety of different cells); or can includevarious combinations of the above described aspects. Accordingly, thepresent invention also provides an agent that modulates reversetranscriptase activity in a cell, such an agent being identifiedaccording to a method as disclosed herein.

The present invention also relates to a method of modulating reversetranscriptase activity in a cell. Such a method can be performed, forexample, by contacting the cell with an agent that alters manganese iontransport across a cell membrane of the cell, thereby modulating reversetranscriptase activity in the cell. The agent can be one that reduces orinhibits manganese ion transport out of the cell. In one aspect, theagent is one that reduces or inhibits manganese ion transport across acell membrane of the cell, but that does not alter the transport ofother divalent cations across the cell membrane. In another aspect, theagent is one alters the activity of a divalent cation transportingprotein in a cell membrane of the cell, for example, a P-type ATPasesuch as a Pmr1p protein or a homolog thereof.

The present invention further relates to a method of modulating reversetranscriptase activity by contacting the reverse transcriptase, underconditions suitable for reverse transcriptase activity, with apredetermined concentration of manganese ions. The conditions can be anyconditions suitable for reverse transcriptase activity, including, forexample, an in vitro reaction mixture containing a buffer,deoxyribonucleotide triphosphates, and/or a primer, and/or can includean extract of a cell, for example, an extract of a cell infected with aretrovirus. In one aspect, the method further includes contacting thereverse transcriptase with a predetermined concentration of magnesiumions. As such, the method provides a means to modulate the relativeactivity of a reverse transcriptase with respect to a polyribonucleotidetemplate as compared to a polydeoxyribonucleotide template.

The present invention also relates to a method of ameliorating aretrovirus infection in a subject. Such a method can be performed, forexample, by contacting cells of the subject with an agent that altersmanganese ion transport in a retrovirus infected cell of the subject.The cells can be contacted with the agent in vivo, for example, byadministering the agent systemically to the subject such that the agentcirculates to the retrovirus infected cells, or by administering theagent at or near the site of the retrovirus infected cells in thesubject. Alternatively, the cells can be contacted with the agent exvivo, after which the cells can be expanded in culture without concernfor replication of the retrovirus due to inhibition of the retrovirusreverse transcriptase, and uninfected cells of the expanded populationcan be selected and administered back into the subject. Preferably, theagent is one that reduces or inhibits a divalent cation transportingprotein activity in the retrovirus infected cell, and more preferably,the agent does not alter transport of a divalent cation other than amanganese ion by the divalent cation transporting protein. As such, amethod of the invention can be useful, for example, for treating a humaninfected with HIV-1, or for treating poultry infected with avianmyeloblastosis virus, and can further be useful, for example, fortreating cats infected with feline leukemia virus, and the like.

The present invention further relates to a high throughput assay foridentifying an agent that alters manganese ion transport by a divalentcation transporting protein in a cell. Such a method can be performed,for example, by providing an array, which can be an addressable array,containing a plurality of cells, including one or more cells atpositions of the array, that express a heterologous divalent cationtransporting protein, wherein the heterologous divalent cationtransporting protein transports at least manganese ion. For example, theplurality of cells can be a plurality of yeast cells, insect cells,avian cell, mammalian cells, or combinations thereof. The heterologousdivalent cation transporting protein can be any transporter that isheterologous with respect to the cell containing the transporter (i.e.,any transporter that is not expressed in the particular cell in nature,or that is expressed from a recombinant nucleic acid molecule introducedinto the cell). In one embodiment, the cell is a yeast cell, and theheterologous transporter is a human divalent cation transporting proteinexpressed in the yeast cell. In one aspect of this embodiment, the humandivalent cation transporting protein comprises a human Pmrp1transporting protein.

A support comprising the array can be any support suitable forcontaining cells of the plurality being contacted with an agent (orotherwise in the array) in relative isolation from other cells of theplurality. For example, the support can be a microchip, wherein cells ofthe plurality can be positioned on the surface of the microchip,including, for example, in a depression or other delimited area of themicrochip. The support also can comprise an array of wells, for example,as provided in a microtiter plate or the like, which can contain 8wells, 24 wells, 96 wells, 384 wells, 1092 wells, or any number of wellsas desired, and can be composed of any suitable material that is nottoxic to the cells to be contacted with the wells and that does notsubstantially react with reagents to be contacted with the cells in thewell, including, for example, test agents to be added to the wells.

The cells of the plurality can further express a reporter gene,particularly wherein expression of the reporter gene is regulateddirectly or indirectly by manganese ion concentration in the cell. Inone embodiment, the reporter gene is a gene regulated by reversetranscriptase activity in the cell. In one aspect of this embodiment,the reporter gene comprises a retrotransposable element, whereinexpression of the reporter gene comprises detecting integration (or alack thereof) of the element into the genome of the cell. In anotherembodiment, the reporter gene comprises is a hybrid Ty-HIV (HART)reporter construct, which is useful for detecting reverse transcriptaseactivity.

The present invention also relates to a method of identifying an agentthat modulates reverse transcriptase activity in a cell. Such a methodcan be performed, for example, by contacting cells of an array of cellswith at least one test agent, wherein the cells comprise a divalentcation transporting protein, which transports manganese ions; anddetecting altered manganese ion transport in cells of the array due tocontact with the test agent as compared to manganese ion transport inthe absence of the test agent, thereby identifying an agent thatmodulates reverse transcriptase activity in a cell. The cells of thearray can be eukaryotic cells, for example, yeast cells or mammaliancells. Further, the divalent cation transporting protein expressed bythe cells of the array can be an endogenous transporter, or can be aheterologous divalent cation transporting protein. In one embodiment,the method is performed using an array of yeast cells. In one aspect ofthis embodiment, the heterologous divalent cation transporting proteincomprises a human divalent cation transporting protein. In anotheraspect of this embodiment, the heterologous divalent cation transportingprotein comprises a human Pmrp1 transporting protein.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 illustrates a proposed two metal ion mechanism of DNApolymerization for Ty1/HIV-1 reverse transcriptase (RT) based on thecrystal structures of mammalian DNA polymerase β (Pelletier et al.,Science 264: 1891-1903, 1994; Sawaya, Science 264: 1930-1935, 1994;Sawaya et al., Biochemistry 36: 11205-11215, 1997), Taq DNA polymerase(Li et al., EMBO J. 17: 7514-7525, 1998), T7 DNA polymerase (Doublie etal., Nature 391, 251-8, 1998), and HIV-1 RT (Huang et al., Science 282,1669-1675, 1998)—modified from Brautigam and Steitz, Curr. Opin. Struct.Biol. 8: 54-63, 1998. Aspartic acid-129 (D129) and D210 for Ty1, andD110 and D185 for HUV-1, represent the two active site aspartates thatare conserved among DNA polymerases, while D211 for Ty1 and D186represent the third active site aspartate that is conserved amongreverse transcriptases.

FIG. 2 illustrates the PMR1 locus, the insert contained within c24-2 andsubsequent subclones (see Example 1). Open boxes represent genestranscribed from left to right, and gray boxes represent genestranscribed from right to left. Coordinates (base pairs; bp) are thoseassigned by SGD (see world wide web, hypertext transport protocol“genome-www.stanford.edu/Saccharomyces/) for chromosome seven.

FIGS. 3A and 3B show a characterization of VLPs isolated from wild-type(PMR1) and pmr1Δ cells. Quantitative immunoblotting of sucrose gradientfractions was performed using anti-integrase, anti-reversetranscriptase, and anti-Gag antibodies.

FIG. 3A shows the VLP-associated RT activity in 10 mM MgCl₂ for theindicated gradient fractions. RT activity of VLPs isolated fromwild-type (PMR1) cells (black circles with solid black line) and pmr1Δcells (gray circles with dashed gray line). RT activity of wild-typeVLPs was adjusted to normalize for slight differences in the amount ofRT protein.

FIG. 3B shows the Mg²⁺-dependent (circles) and Mn²⁺-dependent(triangles) RT activity of VLPs isolated from wild-type cells (solidblack lines) and pmr1Δ cells (dashed gray lines). 100% incorporationcorresponds to 2 pmol dGTP incorporated/μl of VLPs (fraction 24).

FIG. 4 illustrates Mn²⁺-dependent inhibition of RNA-directed DNApolymerization. Reactions were performed with 10 mM (circles), 5 mM(triangles), 3 mM (squares), or 2 mM (diamonds) MgCl₂ and varyingconcentrations (0.2 μM-20 mM) of MnCl₂ (black and red lines) or CaCl₂(green line). VLPs were isolated from wild-type cells (black lines) andpmr1Δ cells (gray lines). 100% incorporation corresponds to 5 pmol dGTPincorporated/μl of VLPs, 137 pmol dGTP incorporated/μg Ty1 RT, and 205pmol dGTP incorporated/μg HIV-1 RT during the 60 min RT assays. Straightdashed gray lines indicate half-maximal incorporation conditions.

FIG. 4A shows metal ion competition for Ty1 VLPs.

FIG. 4B shows metal ion competition for hetero-dimeric HIV-1.

FIG. 4C shows metal ion competition for wild-type Ty1 RT.

FIG. 4D shows wild-type (black) compared to D211N (red) Ty1 RT.

FIG. 5 shows metal-dependent activation of purified recombinant Ty1 RTduring RNA-directed DNA polymerization. Specific activity of wild-typeRT as a function of total MgCl₂ (FIG. 5A) or MnCl₂ (FIG. 5C). Specificactivity of D211N RT as a function of total MgCl₂ (FIG. 5B) and MnCl₂(FIG. 5D). Results are a combination of at least three independentexperiments. 100% incorporation corresponds to 137 pmol dGTPincorporated/μg RT during the 60 min assays.

FIG. 5E shows the activity of wild-type RT (black) and D211N RT (red)for MgCl₂ (squares) and MnCl₂ (diamonds) represented on a Hill plot.Under the experimental conditions, more than 94% of the divalent cationconcentration was free, consistent with the Hill equation (i.e.,log(v/(V_(Max)−v))=n_(H)log{M}+log K_(0.5); where v=velocity,V_(Max)=maximal velocity, n_(H)=Hill coefficient, {M}=divalent cationconcentration, and K_(0.5)=macroscopic equilibrium constant for thedivalent cation). Solid lines correspond to results of linear regressionanalysis of each data set. Slopes of these lines are given as Hillcoefficients in Table 1.

DETAILED DESCRIPTION OF THE INVENTION

The present invention provides methods for identifying an agent thatmodulates the activity of reverse transcriptase. The methods of theinvention are based on the demonstrated ability of the concentrations ofcertain divalent cations to affect the activity of reverse transcriptasein a cell. The validity of a method of the invention is exemplified bythe identification of functional mutations to a divalent cationtransporting protein that is localized within a membrane of a yeast celland is responsible for the regulation of intracellular divalent cationconcentrations. Specifically, mutations to a transporting protein thatresulted in altered transport of divalent cation, manganese ion,significantly modulated reverse transcriptase activity. Accordingly, thepresent invention provides methods of identifying an agent thatmodulates reverse transcriptase activity, and further provides methodsof modulating reverse transcriptase activity, and methods ofameliorating a retrovirus infection in a subject.

Reverse transcriptase (RT) is an enzyme that can construct doublestranded DNA molecules from a single stranded polynucleotide template,such as the RNA template of a retrovirus genome. Although originallydiscovered in retroviruses, RT is encoded by the genomes of a widevariety of retrotransposable elements including, for example,retrotransposons such as Ty elements. All known RTs are multifunctional,having three different enzymatic activities: an RNA-dependent DNApolymerase activity, an RNase H activity, and a DNA-dependent DNApolymerase activity. During retroviral replication RNA-dependent DNApolymerase activity generates a DNA strand (the minus strand)complementary to the viral RNA. This step is followed by the degradationof the original viral RNA strand by RNase H, then the DNA-dependent DNApolymerase activity generates a second DNA strand (the plus strand)complementary to the first. The double stranded DNA is integrated intothe host genome through the action of the retroviral integrase,resulting in a latent infection of the host cell.

The term “retrotransposable element” is used broadly herein to refer toretrotransposons and retroviruses, which are characterized, in part, inthat they encode a RT and an integrase, can integrate into a host cellgenome, and can be maintained in the host cell in a latent form.Retrotransposons are exemplified by copia elements of Drosophila and Tyelements of Saccharomyces cerevisiae. Retrotransposition of a Ty elementis a replicative process involving reverse transcription of Ty mRNA andintegration of Ty cDNA into the genome. Saccharomyces cerevisiae harborsfive types of Ty elements, all of which are long terminal repeat (LTR)retrotransposons, including Ty1, which numbers approximately 30 copiesper haploid genome. Transcription of Ty1 produces a terminally redundantRNA molecule from which the structural protein, Gag, and enzymes, Pol,are translated. Specifically, GAG encodes the capsid (CA) protein andPOL encodes the protease (PR), integrase (IN), and reversetranscriptase/RNase H (RT) (Boeke et al., Cell 40: 491-500, 1985;Garfinkel et al., Cell 42: 507-517, 1985; Mellor et al., Nature 318:583-586, 1985). The Ty1 life cycle can be divided into three phases: 1)expression and assembly, 2) reverse transcription, 3) integration. Gagand Gag-Pol proteins are translated and co-assembled with Ty1 RNA in thecytoplasm, forming virus-like particles (VLPs). VLPs are directtransposition intermediates in which reverse transcription occurs(Eichinger and Boeke, Cell 54: 955-966, 1988; Garfinkel et al., J.Virol. 65: 4573-4581, 1991). Reverse transcription of Ty1, like that ofretroviruses, involves conversion of the terminally redundant RNA into adouble-stranded DNA copy. During this process, the RT uses both RNA andDNA as templates for DNA synthesis. RT activity requires primer,template, and deoxynucleotide triphosphates (dNTPs) as well as adivalent cation, magnesium ion or manganese ion (Wilhelm et al.,Biochem. J. 348: 337-42, 2000, which is incorporated herein byreference; Garfinkel et al., supra, 1985). The contents of theDNA-containing VLPs are transported to the nucleus, where integrationinto host DNA occurs (Kenna et al., Mol. Cell. Biol. 18: 1115-1124,1998; Moore et al., Mol. Cell. Biol. 18: 1105-1114, 1998).

Retroviruses, which can infect fish, amphibian, reptile, bird, andmammalian cells, including, for example, human immunodeficiency virus(HIV), which infects human T lymphocytes, are classified as oncovirusessuch as avian leukemia virus (ALV), Rous sarcoma virus (RSV),Mason-Pfizer monkey virus, and simian retrovirus type 1 and type 2;lentiviruses such as human immunodeficiency virus (HIV) type I (HIV-1)and type II (HIV-2); and spumaviruses. In addition to causing disease,retroviruses have been used as a basis for designing vectors for genetherapy. As discussed above, retroviruses have a two stage life cycle,existing in an RNA form and a DNA form. The RNA form of the virus ispackaged into an infectious particle that is coated with a glycoprotein(env), which is recognized by receptors on the host cell. Thisinteraction promotes a receptor mediated internalization event,resulting in exceptionally efficient delivery of the retroviral genomeinto the cell, where it is converted into a DNA form and can integrateinto the host cell genome.

Reverse transcriptase (RT) activity requires, in addition to the RT, aprimer, a template, and deoxynucleotide triphosphates (dNTPs), as wellas a divalent cation, for example, magnesium or manganese (Garfinkel etal., supra, 1985; Wilhelm et al., supra, 2000). Sequence and structuralcomparisons among reverse transcriptase and DNA polymerases stronglyfavor a nucleotidyl transfer reaction mechanism activated by twodivalent cations sharing a common ligand, as originally found for3′-5′-exonuclease reactions (Beese and Steitz, EMBO J. 10: 25-33, 1991;Han et al., Biochemistry 30: 11104-11108, 1991; see, also, FIG. 1).Crystal structures of mammalian DNA polymerase β (pol β; Pelletier etal., Science 264: 1891-1903, 1994; Sawaya et al., Biochemistry 36:11205-11215, 1997), Thermus aquaticus (Taq) DNA polymerase (Li et al.,EMBO J. 17: 7514-7525, 1998), T7 DNA polymerase (Doublie et al., Nature391: 251-258, 1998) and HIV-1 RT (Huang et al., Science 282: 1669-1675,1998) in primer-template complex with dNTP (the ternary complex)revealed that all of the polymerases bind two divalent cations (normallymagnesium ions) in a binuclear complex at the active site. In thehomologous palm domains, the two metal ions share two completelyconserved aspartate 5 ligands (see FIG. 1; metals A and B). In theternary complexes, metal ion A is proposed to facilitate the attack ofthe 3′ hydroxyl of the primer terminus on the α-phosphorus of the dNTP(Doublie et al., supra, 1998; Steitz, J. Biol. Chem. 274: 17395-17398,1999). Both metal ions are hypothesized to stabilize the pentacovalenttransition state, while metal ion B is proposed to facilitate theleaving of pyrophosphate (Id). However, among RTs, a third conservedaspartate residue (see FIG. 1) has been positioned at the active sitesof HIV-1 RT structurally (Huang et al., Science 282: 1669-1675, 1998)and Ty1 RT functionally (Uzun and Gabriel, J. Virol. 75: 6337-6347,2001, which is incorporated herein by reference).

As disclosed herein, manipulation of manganese ions in a cell canmodulate RT activity, including reducing or inhibiting RT activity. Asused herein, the term “reverse transcriptase activity” refers to thepolymerase and/or RNAase H activity of a RT, including the ability toeffect the formation of a polydeoxyribonucleotide sequence using an RNAtemplate or a DNA template, and/or the ability to degrade an RNAcomponent of a DNA/RNA hybrid. RT activity can be measured using anyassay as disclosed herein or otherwise known in the art. For example, RTactivity can be measured in a yeast cell based assay using a Ty-HIV-1(HART) reporter construct, which includes domains of TY-1, His3AI andthe RT/RNAse H domain of human HIV-1 (Nissley et al., Nature 380: 30,1996; Nissley et al., Proc. Natl. Acad. Sci., USA 95: 13905-13910, 1998,each of which is incorporated herein by reference).

The term “modulate”, when used in reference to RT activity, means thatthe RT activity is increased, or is reduced or inhibited, as compared toa control level. Generally, the control level is the RT activity underdefined conditions in the absence of contact with an ant that altersmanganese ion transport. The terms “reduce or inhibit” are used togetherherein because it is recognized that, depending on a particular assaybeing used, the level of RT activity can be reduced below a level thatcan be detected using the assay and, therefore, it may not always beclear whether the RT activity is completely inhibited. Similarly, theterm “alter”, when used in reference to manganese transport or todivalent cation transporting protein activity, means that the level ofsuch transport or activity is increased, or is reduced or inhibited,with respect to a control level of activity. As such, the terms“modulate” and “alter” can be used interchangeably.

A method of the invention can be performed, for example, by contacting acell membrane, which contains a divalent cation transporting proteinthat transports manganese ions, and can transport other divalentcations, with a test agent; and detecting altered manganese iontransport due to contact with the test agent as compared to manganeseion transport in the absence of the test agent. The term “cell membrane”is used broadly herein to refer to any membrane normally associated witha cell, particularly a eukaryotic cell. As such, a cell membrane usefulin a method of the invention generally comprises a lipid bilayer and isexemplified by a cell surface membrane, which defines an intracellularcompartment and an extracellular compartment, and by membranesassociated with an organelle of a eukaryotic cell, for example, anuclear membrane, Golgi apparatus membrane, mitochondrial membrane, andendoplasmic reticulum membrane.

The cell membrane contacted with the test agent can be an isolated cellmembrane, which contains the divalent cation transporting protein, forexample, a eukaryotic cell membrane such as a yeast cell membrane or amammalian cell membrane (e.g., a human cell membrane), and can be anycell membrane of a cell, including, for example, the cell surfacemembrane, or a cell membrane of associated with the Golgi apparatus,mitochondria, endoplasmic reticulum, or nucleus. Alternatively, the cellmembrane contacted with the test agent can be a cell membrane in situ,in which case the method is performed by contacting a cell, whichcomprises the cell membrane. The cell can be any type of cell thatcontains a cell membrane with a divalent cation transporting protein andthat supports RT activity. As such, the cell can be a eukaryotic cell,including, for example, an insect cell (e.g., a Drosophila cell), afungus cell (e.g., a Neurospora cell), a yeast cell, a C. elegans cell,an amphibian cell (e.g., sea urchin), an avian cell (e.g., a chickembryo fibroblast), or a human cell (e.g., a human T lymphocyte).Further, such cells useful in a method of the invention can be cells ofa cell line, which have been adapted to culture; can be cells of aprimary cell culture, which can be maintained in culture for at least ashort period of time; or cells that have been isolated from a livingorganism, for example, cells isolated from a human subject.

A characteristic of a cell membrane useful in a method of the inventionis that the it contains a divalent cation transporting protein. As usedherein, the term “divalent cation transporting protein” or “divalentcation transporter” refers to a cell membrane-associated structure thatis involved in the transport of divalent cations in one or bothdirections across the cell membrane. As such, it should be recognizedthat, for purposes of the present invention, a cell membrane generallyis substantially impermeable to cations, particularly manganese and anyother cation or cations that are transported by the particular divalentcation transporter being examined according to a screening method asdisclosed herein. Divalent cation transporting proteins include ionchannels, molecular transporters and ion pumps, and, in addition tomanganese, divalent cations transported by such transporting proteinscan include, for example, magnesium and calcium.

Ion channels are typically formed by the association of integralmembrane proteins into structures having a central hydrophilic pore.Channel pores allow ions to equilibrate across membranes in response totheir electrochemical gradients and at rates that are diffusion limited.Ion channels are characterized by their selectivity and gatingproperties. Selectivity refers to the rate at which different ionspecies pass through an open channel under standard conditions. Gatingis the process that regulates the opening and closing of an ion channel.Thus, voltage-regulated ion channels respond to changes in membranepotential; ligand-regulated channels respond to the binding ofparticular ligands or intracellular messengers (e.g., cyclicnucleotides, calcium ions); and mechanosensitive channels respond tomechanical deformation (e.g., stretch).

Ion channels exist in resting (closed), open or inactivated (i.e.,desensitized) states. Voltage-gated ion channels in the open statetypically transition to an inactivated state, and must reacquire theability to respond to an external stimulus during a recovery period.This may also be true of ligand-gated channels, particularly afterprolonged exposure to an agonist. Certain channels are gated by morethan one type of stimulus (e.g., an inward rectifying voltage-regulatedpotassium channel in cardiac muscle is activated by acetylcholine). Ionchannels serve a variety of important cellular functions, includingstimulation, excitability, signaling, excitation-secretion coupling,volume regulation and so on. Ion channels are implicated in a variety ofpathophysiological disorders, including hypertension, cardiacarrhythmogenesis, non-insulin dependent diabetes mellitus, and seizures,and mediate the transmission of pain impulses by peripheral nerves (see,generally, Ackerman and Clapham, New Engl. J. Med. 336: 1575, 1997).

The ABC transporters comprise a superfamily that shares a highlyconserved ATP-binding cassette (Higgins, Ann. Rev. Cell Biol. 8: 67-113,1992). These transporters typically use ATP hydrolysis as a source ofenergy to pump diverse classes of molecules (e.g., sugars, peptides,inorganic ions, amino acids, oligopeptides, polysaccharides, proteins)across membranes against a concentration gradient. Each transporter ishighly selective for a particular substrate and pumps unidirectionally.Some members of the ABC transporter family have ion channel activity.For example, the cystic fibrosis transmembrane regulator (CFTR), a cAMPand protein kinase A regulated chloride ion channel, uses ATP hydrolysisas a gating mechanism. P-glycoprotein (MDR) appears to be bifunctional,possessing drug transport as well as chloride channel activities; thelatter is cell-volume regulated and requires the binding, but not thehydrolysis, of ATP. In both prokaryotes and eukaryotes, ABC transportersfunction in nutrient uptake, protein export and drug resistance (e.g.,erythromycin resistance in Staphylococcus, daunomycin resistance inStreptomyces, chloroquine resistance in Plasmodium, and multidrugresistance in cancers).

Ion pumps are also involved in the active transport of ions acrossmembranes. Ion pumps are members of the ion-transporting P-type ATPasefamily, which couple ion transport to a cycle of phosphorylation anddephosphorylation of an ATPase enzyme. In mammalian cells, this classincludes the calcium ion ATPases, the sodium ion/potassium ion ATPases,and the hydrogen ion/potassium ion ATPases, the latter of which areinvolved in acid secretion in the stomach and are clinically importanttargets in peptic ulcer disease, gastroesophageal reflux disease (GERD)and gastric hyperacidity. The calcium ion ATPases and sodiumion/potassium ion ATPases, in comparison, are therapeutic targets in thetreatment of heart failure.

A divalent cation transporting protein examined according to a method ofthe invention can be any transporter that is involved in the selectivepassage of manganese ions across the cell membrane. Such a transportergenerally, but not necessarily, utilizes an active transport mechanismto pump manganese ions into a cell, out of a cell, or in eitherdirection, depending, for example, on the relative concentration ofmanganese ions in one of the compartments, particularly an intracellularcompartment, as compared to a manganese concentration typically found inthe compartment in an otherwise normal cell. For example, the divalentcation transporting protein can be a P-type ATPase such as an S.cerevisiae Pmr1p protein, which is a calcium ion and manganese iontransporting protein, or a homolog thereof, including for example, ahuman calcium-transporting ATPase type 2C family member (ATP-dependentcalcium ion pump, PMR1; e.g., ATPase 2C1; see GenBank Acc. Nos:AAF26295, AAF26296, and P98194; Hu et al., Nat. Genet. 24: 61-65, 2000;Sudbrak et al., Hum. Mol. Genet. 9: 1131-1140, 2000; Nagase et al., DNARes. 7: 63-73, 2000; Stanchi et al., Yeast 18: 69-80, 2001, each ofwhich is incorporated herein by reference) and isoforms thereof, as wellas the Pmr1p homologs ATP2A1 and ATP2A2, which are expressed in cardiaccells, and ATP2A3, which is expressed ubiquitously, sequences of whichcan be obtained by a search, on the world wide web, at the URL“ncbi.nlm.nih.gov”, in the Entrez Protein database.

Generally, an isolated cell membrane useful in a method of the inventiondelimits at least a first compartment and a second compartment, whereinthe divalent cation transporting protein in the membrane can transportdivalent cations, including manganese, from the first compartment to thesecond, or from the second compartment to the first; or can transportdivalent cations into and out of both compartments, includingtransporting manganese into and/or out of at least one compartment. Forexample, a portion of a cell membrane can be obtained using amicrocapillary, wherein the cell membrane can separate the contents ofthe microcapillary (i.e., a first compartment) from a medium in whichthe microcapillary is contacted (i.e., a second compartment). Accordingto such an example, the test agent can be present in or added to themedium (second compartment), manganese ions can be present in the firstand/or second compartment, and altered manganese ion transport can bedetected by examining the first compartment or the second compartment orboth.

Altered manganese ion transport can be detected using any method asdisclosed herein or otherwise known in the art. For example, alteredmanganese ion transport can be detected by detecting movement ofmanganese ions from one compartment to another, by detecting a change inthe concentration of manganese ions in one or both compartments, or bydetecting a change in a functional activity associated with a change inmanganese ion concentration in a compartment, for example, as disclosedherein, by detecting a change in RT activity in compartment. Forexample, altered manganese ion transport can be detected using a patchclamp method. The patch clamp technique is commonly used for examiningtransmembrane proteins, and can provide a “voltage clamp” measurement ofionic current in either a small “patch” of cell membrane, or the entiremembrane of a small cell. Because the method measures current, itdirectly monitors the number of active channels in the membrane and,therefore, can be an appropriate assay for identifying agents that blockor otherwise modulate divalent cation transporter activity (see,generally, Boulton et al. (eds.), Patch Clamp Applications andProtocols, Humana Press (1995); Neher and Sakmann (eds.), Single-ChannelRecording, Plenum Press (1995), and DeFelice, Electrical Properties ofCells: Patch Clamp for Biologists (The Language of Science), Plenum Pub.Corp. (1997), each of which is specifically incorporated by reference inits entirety). Altered magnesium ion transport also can be detectedusing a polarographic (voltametric) method or a radiometric method, inwhich the concentration of manganese ions can be determined.

Altered manganese ion transport due to contact of a cell membrane with atest agent also can be detected by detecting a change in RT activity ina relevant compartment, which can be in intracellular compartment orother compartment delimited by the cell membrane. RT activity can bemeasured using a polynucleotide template, which can be a DNA or an RNAtemplate, or both. The RT being detected can be an RT that is present ina cell due, for example, to infection of the cell by a retrotransposableelement that expresses the RT, or to expression in a cell of anexogenously added nucleic acid molecule encoding the RT, or can be thatof an isolated RT polypeptide.

A method of the invention provides a means to examine test agents toidentify those agents that can to modulate RT activity and/or alterdivalent cation transporter activity. The term “test agent” is usedherein to mean any compound that it to be examined for an ability tomodulate RT activity and/or alter divalent cation (particularlymanganese ion) transporter activity using a screening assay of theinvention. A test agent can be a compound that is known to have such anability, wherein the screening assay is used to confirm the activity,for example, with respect to a different transporter protein than one itis known to be able to alter; or to determine an amount of the agentthat can be useful for modulating RT activity in a desired manner, forexample, to reduce or inhibit the RT activity; or for standardizing theactivity of the agent. A test agent also can be compound that is notknown to have such activity and is being tested for such activity, thusproviding a means to identify new agents potentially useful as drugs formodulating RT activity or for treating a disorder associated withundesirable divalent cation transporter activity. Such test agents canbe agents that are based on an agent known to have an ability tomodulate RT activity and/or alter divalent cation (particularlymanganese ion) transporter activity, but that are modified, derivatized,or the like.

The term “agent” is used herein to refer to a test that is identified bythe screening assay as having such an ability to modulate RT activityand/or alter divalent cation transporter activity. Such an agent, whichcan modulate RT activity, can be one that alters the transport of allcations that are transported by a particular divalent cationtransporting protein, or, in particular, an agent that only altersmanganese ion transport, but not any other divalent cations (if any)that can be transported by the divalent cation transporting protein.Further, the method can be used to identify an agent that reduces orinhibits manganese ion transport out of a cell, or that increasesmanganese transport into a cell, thus providing an agent that canincrease an intracellular manganese ion concentration in a cell above alevel normally found in the cell; or the method can be used to identifyan agent that reduces or inhibits manganese ion transport into a cell,or that increase manganese ion transport out of a cell, thus providingan agent that can decrease an intracellular manganese ion concentrationin a cell below a level normally found in the cell.

A test agent can be any type of molecule, for example, a polynucleotide,a peptide a peptidomimetic, peptoids such as vinylogous peptoids, asmall organic molecule, or the like Polynucleotides, for example, areknown to specifically interact with proteins and, therefore, can beuseful as test agents to be screened for the ability to alter theactivity of a divalent cation transporter. The term “polynucleotide” isused broadly herein to mean a sequence of two or moredeoxyribonucleotides or ribonucleotides that are linked together by aphosphodiester bond. As such, the term “polynucleotide” includes RNA andDNA, which can be a synthetic RNA or DNA sequence, and can be singlestranded or double stranded, as well as a DNA/RNA hybrid. Furthermore,the term “polynucleotide” as used herein includes naturally occurringnucleic acid molecules, which can be isolated from a cell, as well assynthetic molecules, which can be prepared, for example, by methods ofchemical synthesis or by enzymatic methods such as by the polymerasechain reaction (PCR). In various embodiments, a polynucleotide useful asa test agent can contain nucleoside or nucleotide analogs, or a backbonebond other than a phosphodiester bond (see above).

In general, the nucleotides comprising a polynucleotide are naturallyoccurring deoxyribonucleotides, such as adenine, cytosine, guanine orthymine linked to 2′-deoxyribose, or ribonucleotides such as adenine,cytosine, guanine or uracil linked to ribose. However, a polynucleotidealso can contain nucleotide analogs, including non-naturally occurringsynthetic nucleotides or modified naturally occurring nucleotides. Suchnucleotide analogs are well known in the art and commercially available,as are polynucleotides containing such nucleotide analogs (Lin et al.,Nucl. Acids Res. 22: 5220-5234, 1994; Jellinek et al., Biochemistry 34:11363-11372, 1995; Pagratis et al., Nature Biotechnol. 15: 68-73, 1997,each of which is incorporated herein by reference).

The covalent bond linking the nucleotides of a polynucleotide generallyis a phosphodiester bond. However, the covalent bond also can be any ofnumerous other bonds, including a thiodiester bond, a phosphorothioatebond, a peptide-like bond or any other bond known to those in the art asuseful for linking nucleotides to produce synthetic polynucleotides(see, for example, Tam et al., Nucl. Acids Res. 22: 977-986, 1994; Eckerand Crooke, BioTechnology 13: 351360, 1995, each of which isincorporated herein by reference). The incorporation of non-naturallyoccurring nucleotide analogs or bonds linking the nucleotides or analogscan be particularly useful where the polynucleotide is to be exposed toan environment that can contain a nucleolytic activity, including, forexample, a tissue culture medium or upon administration to a livingsubject, since the modified polynucleotides can be less susceptible todegradation.

A polynucleotide comprising naturally occurring nucleotides andphosphodiester bonds can be chemically synthesized or can be producedusing recombinant DNA methods, using an appropriate polynucleotide as atemplate. In comparison, a polynucleotide comprising nucleotide analogsor covalent bonds other than phosphodiester bonds generally will bechemically synthesized, although an enzyme such as T7 polymerase canincorporate certain types of nucleotide analogs into a polynucleotideand, therefore, can be used to produce such a polynucleotiderecombinantly from an appropriate template (Jellinek et al., supra,1995).

A peptide also can be useful as an agent that alters divalent cationtransporter activity. The term “peptide” is used broadly herein to meantwo or more amino acids linked by a peptide bond. Generally, a peptideuseful in a method of the invention contains at least about two, three,four, five, or six amino acids, and can contain about ten, fifteen,twenty or more amino acids. As such, it should be recognized that theterm “peptide” is not used herein to suggest a particular size or numberof amino acids comprising the molecule, and that a peptide of theinvention can contain up to several amino acid residues or more.Generally, however, smaller peptides are preferred where an identifiedagent is to be further examined, for example, for use as a drug fortreating a subject. A peptide test agent can be prepared, for example,by a method of chemical synthesis, or can be expressed from apolynucleotide using recombinant DNA methodology. Where chemicallysynthesized, peptides containing one or more D-amino acids, or one ormore amino acid analogs, for example, an amino acid that has beenderivatized or otherwise modified at its reactive side chain, or inwhich one or more bonds linking the amino acids or amino acid analogs ismodified, can be prepared. In addition, a reactive group at the aminoterminus or the carboxy terminus or both can be modified. Such peptidescan be modified, for example, to have improved stability to a protease,an oxidizing agent or other reactive material the peptide may encounterin a biological environment, and, therefore, can be particularly usefulin performing a method of the invention. Of course, the peptides can bemodified to have decreased stability in a biological environment suchthat the period of time the peptide is active in the environment isreduced.

As disclosed herein, the screening methods of the invention provide theadvantage that they can be adapted to high throughput analysis and,therefore, can be used to screen combinatorial libraries of test agentsin order to identify those agents that can alter divalent cationtransporting protein activity. Methods for preparing a combinatoriallibrary of molecules that can be tested for a desired activity are wellknown in the art and include, for example, methods of making a phagedisplay library of peptides, which can be constrained peptides (see, forexample, U.S. Pat. Nos. 5,622,699; 5,206,347; Scott and Smith, Science249: 386-390, 1992; Markland et al., Gene 109: 13-19, 1991; each ofwhich is incorporated herein by reference); a peptide library (U.S. Pat.No. 5,264,563, which is incorporated herein by reference); apeptidomimetic library (Blondelle et al., Trends Anal. Chem. 14: 83-92,1995; a nucleic acid library (O'Connell et al., Proc. Natl. Acad. Sci.,USA 93: 5883-5887, 1996; Tuerk and Gold, Science 249: 505-510, 1990;Gold et al., Ann. Rev. Biochem. 64: 763-797, 1995; each of which isincorporated herein by reference); an oligosaccharide library (York etal., Carb. Res., 285: 99-128, 1996; Liang et al., Science, 274:1520-1522, 1996; Ding et al., Adv. Expt. Med. Biol. 376: 261-269, 1995;each of which is incorporated herein by reference); a lipoproteinlibrary (de Kruif et al., FEBS Lett. 399: 232-236, 1996, which isincorporated herein by reference); a glycoprotein or glycolipid library(Karaoglu et al., J. Cell Biol. 130: 567-577, 1995, which isincorporated herein by reference); or a chemical library containing, forexample, drugs or other pharmaceutical agents (Gordon et al., J. Med.Chem. 37: 1385-1401, 1994; Ecker and Crooke, BioTechnology 13: 351-360,1995; each of which is incorporated herein by reference).Polynucleotides can be particularly useful as agents that can modulate aspecific interaction of myostatin and its receptor because nucleic acidmolecules having binding specificity for cellular targets, includingcellular polypeptides, exist naturally, and because synthetic moleculeshaving such specificity can be readily prepared and identified (see, forexample, U.S. Pat. No. 5,750,342, which is incorporated herein byreference).

In performing a screening assay of the invention in a high throughputformat, isolated cell membranes or intact cells can be used. Anadvantage of using intact cells is that the method can be used, forexample, to identify an agent useful for modulating RT activity inparticular cells or cell types by altering manganese ion transport. Forexample, a plurality of human T lymphocytes isolated from a subjectinfected with HIV can be arranged in an array, which can be anaddressable array, on a solid support such as a microchip, on a glassslide, on a bead, or in a well, and the cells can be contacted withdifferent test agents to identify one or more agents having desirablecharacteristics, including, for example, in addition to the ability toalter manganese ion transport, minimal or no toxicity to the cell,desirable solubility characteristics, and the like. An additionaladvantage of arranging the samples in an array, particularly anaddressable array, is that an automated system can be used for adding orremoving reagents from one or more of the samples at various times, orfor adding different reagents to particular samples. In addition to theconvenience of examining multiple samples at the same time, such highthroughput assays provide a means for examining duplicate, triplicate,or more aliquots of a single sample, thus increasing the validity of theresults obtained, and for examining control samples under the sameconditions as the test samples, thus providing an internal standard forcomparing results from different assays.

Accordingly, the present invention also provides methods of modulatingRT activity in a cell. Such a method can be performed, for example, bycontacting the cell with an agent that alters manganese ion transportacross a cell membrane of the cell, thereby modulating RT activity inthe cell. The agent can be one that reduces or inhibits manganese iontransport out of the cell, particularly an agent that reduces orinhibits manganese ion transport across a cell membrane of the cell, butdoes not alter the transport of other divalent cations across the cellmembrane. In addition, the invention provides methods of modulating RTactivity by contacting the RT, under conditions suitable for reversetranscriptase activity, with a predetermined concentration of manganeseions. The conditions can be any conditions suitable for RT activity,including, for example, an in vitro reaction mixture containing abuffer, deoxyribonucleotide triphosphates, and/or a primer, and/or caninclude an extract of a cell, for example, an extract of a cell infectedwith a retrovirus. In one aspect, the method further includes contactingthe RT with a predetermined concentration of magnesium ions, wherein therelative activity of a RT with respect to an RNA template as compared toa DNA template can be manipulated. Such a method can be useful, forexample, for preferentially generating one strand of double stranded DNAcorresponding to a retrovirus genome (i.e., the minus strand, in whichcase the RT is modulated such that it preferentially uses an RNAtemplate, or the plus strand, in which case the RT is modulated suchthat it preferentially uses the minus strand as a template).

The present invention also relates to a method of ameliorating aretrovirus infection in a subject. Such a method can be performed, forexample, by contacting cells of the subject with an agent that altersmanganese ion transport in a retrovirus infected cell of the subject,thereby modulating (e.g., reducing or inhibiting) RT activity in thecells. The cells can be contacted with the agent in vivo, for example,by administering the agent systemically to the subject such that theagent circulates to the retrovirus infected cells, or by administeringthe agent at or near the site of the retrovirus infected cells in thesubject. Alternatively, the cells can be contacted with the agent exvivo, after which the cells can be expanded in culture without concernfor replication of the retrovirus due to inhibition of the retrovirusreverse transcriptase, and uninfected cells of the expanded populationcan be selected and administered back into the subject. Preferably, theagent is one that reduces or inhibits a divalent cation transportingprotein activity in the retrovirus infected cell, and more preferably,the agent does not alter transport of a divalent cation other than amanganese ion by the divalent cation transporting protein.

A subject to be treated according to a method of the invention can beany subject that is susceptible to infection with a retrovirus, andparticularly a subject in the which the infection has a deleteriouseffect. In particular, the subject can be a human or other mammaliansubject in which the retroviral infection, for example, adverselydisrupts a gene in the genome of cells of the subject due to randomintegration into the genome, thus resulting in a loss of function of thegene; or integrates into a gene in the genome of cells and adverselyincreases the expression of the gene, thereby resulting in increasedexpression of all or a portion of the encoded gene product (e.g., anactivated oncogene); or otherwise results in a pathologic condition,such as infection with HIV-1, which can cause AIDS. The subject to betreated according to a method of the invention also can be domesticatedor farm animal, or the like, for example, poultry infected with avianmyeloblastosis virus, or cats infected with feline leukemia virus.

For administration to a subject, an agent that modulates RT activity,particularly by altering manganese ion levels in cells infected with theretrovirus is administered by a route and under conditions thatfacilitate contact of the agent with the target cell and, ifappropriate, entry into the cell. Thus, the agent can be administered tothe site of the cells to be treated, or can be administered by anymethod that provides the target cells with the agent. Furthermore, theagent generally is formulated in a composition (e.g., a pharmaceuticalcomposition) suitable for administration to the subject. As such, theinvention provides pharmaceutical compositions containing an agent,which is useful for altering manganese transport in a cell, in apharmaceutically acceptable carrier. As such, the agents are useful asmedicaments for treating a subject suffering from a pathologicalcondition as defined herein. Further, such a composition can include oneor more other compounds that, alone or in combination with the agentthat manganese transport, provides a therapeutic advantage to thesubject, for example, an antibiotic if the subject is susceptible to abacterial infection, one or more additional antiviral agents known to beuseful for treating the retrovirus infecting the cells of the subject, anutrient or vitamin or the like, a diagnostic reagent, toxin, atherapeutic agent such as a cancer chemotherapeutic agent, or any othercompound as desired, provided the additional compound(s) does notadversely affect the activity of the agent that alters manganesetransport or, if the compound does affect the activity of the agent,does so in a manner that is predictable and can be accounted for informulating the agent that alters manganese transport.

A composition of the invention generally contains the agent formulatedwith one or more other pharmaceutically acceptable carriers, which arewell known in the art and include, for example, aqueous solutions suchas water or physiologically buffered saline or other solvents orvehicles such as glycols, glycerol, oils such as olive oil or injectableorganic esters. A pharmaceutically acceptable carrier can containphysiologically acceptable compounds that act, for example, to stabilizeor to increase the absorption of the conjugate. Such physiologicallyacceptable compounds include, for example, carbohydrates, such asglucose, sucrose or dextrans, antioxidants, such as ascorbic acid orglutathione, chelating agents, low molecular weight proteins or otherstabilizers or excipients. One skilled in the art would know that thechoice of a pharmaceutically acceptable carrier, including aphysiologically acceptable compound, depends, for example, on thephysico-chemical characteristics of the agent that alters manganesetransport and on the route of administration of the composition, whichcan be, for example, orally or parenterally such as intravenously, andby injection, intubation, or other such method known in the art.

The agent that modulates RT activity by altering manganese transport incells infected with a retrovirus can be incorporated within anencapsulating material such as into an oil-in-water emulsion, amicroemulsion, micelle, mixed micelle, liposome, microsphere or otherpolymer matrix (see, for example, Gregoriadis, Liposome Technology, Vol.1 (CRC Press, Boca Raton, Fla. 1984); Fraley et al., Trends Biochem.Sci. 6: 77, 1981, each of which is incorporated herein by reference).Liposomes, for example, which consist of phospholipids or other lipids,are nontoxic, physiologically acceptable and metabolizable carriers thatare relatively simple to make and administer. “Stealth” liposomes (see,for example, U.S. Pat. Nos. 5,882,679; 5,395,619; and 5,225,212, each ofwhich is incorporated herein by reference) are an example of suchencapsulating materials particularly useful for preparing a compositionuseful for practicing a method of the invention, and other “masked”liposomes similarly can be used, such liposomes extending the time thatthe therapeutic agent remain in the circulation. Cationic liposomes, forexample, also can be modified with specific receptors or ligands(Morishita et al., J. Clin. Invest. 91: 2580-2585, 1993, which isincorporated herein by reference). In addition, a polynucleotide agentcan be introduced into a cell using, for example, adenovirus-polylysineDNA complexes (see, for example, Michael et al., J. Biol. Chem. 268:6866-6869, 1993, which is incorporated herein by reference).

The route of administration of a pharmaceutical composition containingan agent that modulates RT activity will depend, in part, on thechemical structure of the molecule. Polypeptides and polynucleotides,for example, are not particularly useful when administered orallybecause they can be degraded in the digestive tract. However, methodsfor chemically modifying polypeptides, for example, to render them lesssusceptible to degradation by endogenous proteases or more absorbablethrough the alimentary tract are well known (see, for example, Blondelleet al., supra, 1995; Ecker and Crook, supra, 1995). In addition, apeptide agent can be prepared using D-amino acids, or can contain one ormore domains based on peptidomimetics, which are organic molecules thatmimic the structure of peptide domain; or based on a peptoid such as avinylogous peptoid.

A pharmaceutical composition as disclosed herein can be administered toan individual by various routes including, for example, orally orparenterally, such as intravenously, intramuscularly, subcutaneously,intraorbitally, intracapsularly, intraperitoneally, intrarectally,intracistemally or by passive or facilitated absorption through the skinusing, for example, a skin patch or transdermal iontophoresis,respectively. Furthermore, the pharmaceutical composition can beadministered by injection, intubation, orally or topically, the latterof which can be passive, for example, by direct application of anointment, or active, for example, using a nasal spray or inhalant, inwhich case one component of the composition is an appropriatepropellant. A pharmaceutical composition also can be administered to thesite of a pathologic condition, for example, intravenously orintra-arterially into a blood vessel supplying a tissue or organcomprising retrovirus infected cells.

The pharmaceutical composition also can be formulated for oralformulation, such as a tablet, or a solution or suspension form; or cancomprise an admixture with an organic or inorganic carrier or excipientsuitable for enteral or parenteral applications, and can be compounded,for example, with the usual non-toxic, pharmaceutically acceptablecarriers for tablets, pellets, capsules, suppositories, solutions,emulsions, suspensions, or other form suitable for use. The carriers, inaddition to those disclosed above, can include glucose, lactose,mannose, gum acacia, gelatin, mannitol, starch paste, magnesiumtrisilicate, talc, corn starch, keratin, colloidal silica, potatostarch, urea, medium chain length triglycerides, dextrans, and othercarriers suitable for use in manufacturing preparations, in solid,semisolid, or liquid form. In addition auxiliary, stabilizing,thickening or coloring agents and perfumes can be used, for example astabilizing dry agent such as triulose (see, for example, U.S. Pat. No.5,314,695).

The total amount of an agent to be administered in practicing a methodof the invention can be administered to a subject as a single dose,either as a bolus or by infusion over a relatively short period of time,or can be administered using a fractionated treatment protocol, in whichmultiple doses are administered over a prolonged period of time. Anadvantage of using a fractionated method is that, upon normal divisionof a retrovirus infected cell, replication of the retrovirus can bereduced or inhibited due to the presence of the agent. One skilled inthe art would know that the amount of the composition to treat aretrovirus infection in a subject depends on many factors including theage and general health of the subject as well as the route ofadministration and the number of treatments to be administered. In viewof these factors, the skilled artisan would adjust the particular doseas necessary. In general, the formulation of the pharmaceuticalcomposition and the routes and frequency of administration for treatmentof human subjects are determined, initially, using Phase I and Phase IIclinical trials.

The following examples are intended to illustrate but not limit theinvention.

EXAMPLE 1

Strains and media. The strains used in this study were JB740(MATαhis3Δ200Δleu2 1 ura3-167), yEB104A (JB740 with pmr1Δ::hphMX4), YH8(MATαhis3Δ200 leu2Δ1 trp1Δ1 ura3-167; Chapman and Boeke, Cell 65:483492, 1991, which is incorporated herein by reference), which isincorporated herein by reference), YH23 (YH8 transformed with plasmidpX3) (Chapman and Boeke, supra, 1991), KM255 (transposition-deficientmutant of YH23). Media were prepared as described by Sherman et al.(Methods in yeast genetics (Cold Spring Harbor Laboratory Press, 1986),which is incorporated herein by reference. Mn²⁺ hypersensitivity wastested by growth on plates containing synthetic complete (SC) mediumsupplemented with 3 mM MnCl₂ (Wei et al., Biochemistry 38: 14534-14541,1999, which is incorporated herein by reference).

Plasmid constructions. Plasmid c24-2I was generated by ligating the StuI -Avr II fragment (4.3 kb) of c24-2 with the Sma I—Xba I fragment (6.0kb) of pRS415 (LEU2 CEN6; Sikorski and Hieter, Genetics 122: 19-27,1989, which is incorporated herein by reference). To simplify thecloning of the single amino-acid-substitution mutations into the PMR1gene, c24-2L was constructed by removing the Xho I—Nde I fragment (1.2kb) from c24-2I, filling in the ends with Klenow (New England Biolabs;Beverly Mass.), and ligating the blunt ends to recircularize theplasmid. Alanine substitution mutations, D778A and Q783A, were generatedin the native PMR1 gene by replacing the Pst I—Sal I fragment (1.1 kb)of c24-2L with the corresponding fragment harboring the respective Pmr1psubstitution mutation in the YEpHR1 backbone (Wei et al., J. Biol. Chem.275: 23927-23932, 2000, which is incorporated herein by reference). TheD53A substitution mutation was introduced into c24-2L by swapping in theXba I fragment (0.3 kb) containing the Pmr1p substitution mutation inthe YEpHR1 backbone (Wei et al., supra, 1999). Recombinant Ty1 RT(Wilhelm et al., supra, 2000) was PCR amplified and subcloned into theBamH I and Xho I sites of pGEX-4T-3 (Amersham Pharmacia Biotech;Piscataway N.J.). The D211N substitution mutation was generated byreplacing the Sph I-Hind III fragment (0.4 kb) of the wild-type Ty1 RTexpression construct with that of pJEF724 DD-DN (Uzun and Gabriel,supra, 2001).

RNA isolation and blot analysis. Total RNA was isolated from 10 mlcultures of yeast grown in YNB supplemented with 1% casamino acids and1% raffinose at 30° C. for about 6 hr. Glucose (represses) or galactose(induces pGal-Ty1 expression and transposition) was added to 2% andcultures were grown at 22° C. for 42 hr. Strains included YH8transformed with pB656 (pGal vector lacking Ty1 sequence), YH23, KM255and EBX10A-2B. Total RNA was extracted by hot acid phenyl (Collart andOliviero, In Current Protocols in Molecular Biology (eds. Ausubel etal., John Wiley & Sons 1993), which is incorporated herein by reference)and fractionated by denaturing gel electrophoresis as described below.

For quantification of ACT1 and Ty1-TRP1 RNAs, 20 μg total RNA was heatdenatured in sample buffer (55% deionized formamide, MOPS buffer, pH7.0, 5% formaldehyde, 8 mM EDTA) and 0.1% bromophenyl blue) beforeelectrophoresis on 1% agarose gels containing MOPS buffer, pH 7.0 (40 mMMOPS, 10 mM sodium acetate and 1 mM EDTA) and 2% formaldehyde. RNA wastransferred by capillary action and fixed by UV crosslinking to GeneScreen Plus™ filters as described by the manufacturer (NEN Life ScienceProducts, Inc.; Boston Mass.). Membrane-bound RNAs were hybridized toACT1-specific (1.2 kb BamH I—Hind III fragment of pΔ10-AHX3) andTRP1-specific (1 kb BamH I fragment of pX8) DNA probes that wereinternally labeled and purified over G25 Sephadex spin columns. Filterswere exposed to a Molecular Dynamics phosphoimager screen.Quantification of the relative steady-state transcripts (ratio ofTy1-TRP1/ACT1) was done using a STORM Imaging System with ImageQuantv1.11 (Molecular Dynamics) and Microsoft Excel software.

Transposition assay. Yeast transformants containing the URA3 Ty1-TRP1donor plasmid, pX3 (pGal-Ty1 element marked with TRP1; Xu and Boeke.,Proc. Natl. Acad. Sci., USA 84: 8553-8557, 1987, which is incorporatedherein by reference), and any of the LEU2 CEN-based vectors were patchedonto SC medium lacking leucine, tryptophan and uracil (SC-Leu-Trp-Ura)with 2% glucose. After 2-3 days at 30° C., yeast patches were replicaplated to SC-Leu-Ura with 2% galactose and incubated at 22° C. for 3days. The Ty1 donor plasmid was shuffled out by growth on SC-Leu with 2%glucose at 30° C. overnight followed by growth on SC-Leu-Trp containing1 g/L of 5-fluoro-orotic acid (5-FOA) (Boeke et al., Mol. Gen. Genet.197: 345-346, 1984) with 2% glucose at 30° C. for 2 days (selectivegrowth for cells containing a transposed copy of Ty1-TRP1).

Transformants containing the URA3-marked Ty1 donor plasmid,pGTy1-H3-mhis3AI (Curcio and Garfinlcel, Proc. Natl. Acad. Sci., USA 88:936-940, 1991, which is incorporated herein by reference), and any ofthe LEU2 CEN-based vectors were spotted onto SC-Leu-Ura with 2% glucose.After 2 days at 30° C., yeast spots were replica plated to similarmedium with 2% galactose and incubated at 22° C. for 3 days. At thispoint Ty1 transposition was assayed by using either single-step(non-5-FOA) selection or 5-FOA selection. Yeast spots to be analyzed bysingle-step selection were resuspended in sterile water to an OD₆₀₀=1.0,and 5-fold serial dilutions were spotted onto SC-Leu-His (lackshistidine) with 2% glucose (selective growth for cells containing atransposed copy of the HIS3-marked Ty1 element). In yeast spots to beanalyzed by 5-FOA selection, the Ty1 donor plasmid was shuffled out asdiscussed above, except 5-FOA-containing medium was SC-Leu-His.

cDNA analysis. Lawns of yeast cotransformed with pGTy1-H3-mhis3AI andvarious LEU2 CEN-based vectors were scraped from SC-Leu-Ura plates with2% glucose and resuspended in 10 ml of YNB medium containing 1% casaminoacids and 1% raffinose. Cells were grown at 30° C. for approximately 6hr to exhaust remaining glucose. Galactose was then added to 2% andcultures were incubated at 22° C. for 24 hr. Cells were harvested asdescribed (Lawler et al., J. Virol. 75: 6769-6775, 2001, which isincorporated herein by reference), genomic DNA was isolated (Boeke etal., Cell 40: 491-500, 1985, which is incorporated herein by reference)from 11 ml cultures, then digested with Afl II. DNA fragments werefractionated by agarose gel electrophoresis and transferred to GeneScreen Plus™ filters. Hybridization was measured using a MolecularDynamics phosphoimager. The ratio of HIS3-specific hybridization for theTy1 cDNA fragment (2.2 kb) relative to that of the pGTy1-H3-mhis3AIfragment (14.3 kb) provided a measure of the amount of Ty1 cDNA.

VLP isolation and analysis. Yeast cells harboring pGTy1-H3-mhis3AI weregrown in 2% galactose at 22° C. for approximately 24 hr. Cell pelletsfrom these 0.5 L cultures were resuspended in 5 ml buffer B (Eichingerand Boeke, Cell 54: 955-966, 1988, which is incorporated herein byreference) and VLPs were isolated. VLP-associated RT activity wasmeasured for sucrose gradient fractions 21-28 as described (Eichingerand Boeke, supra, 1988), except that 3 μl of VLPs in buffer B (no EDTA)were used in 30 μl reactions (50 mM HEPES-KOH, pH 7.8, 3 mM DTT, 0.2 μMdGTP, 0.5 μCi of {α³²P}-dGTP, 1 μg/ml oligo(dG)₁₂₋₁₈, 10 μg/ml poly(rC)_(n) and the indicated concentrations of MgCl₂ and/or MnCl₂).Reactions were set up on ice, carried out for 1 hr at 22° C. and kept onice while being spotted onto DE81 anion-exchange paper (WhatmanInternational Ltd.; Maidstone, England).

Immunoblot analysis was performed on gradient fractions to normalize theRT activity of each fraction by the amount of Ty1 RT in the fraction.For detection of IN (integrase), RT and Gag, 2 μl, 2 μl and 0.1 μl,respectively, of each gradient fraction were boiled in SDS-PAGE samplebuffer and run onto 5% stacking/10% separating SDS polyacrylamide gels.Proteins were transferred onto 0.45 μm PVDF membranes (IMMOBILON-P;Millipore Corp.; Bedford Mass.) in transfer buffer (25 mM Tris base, 192mM glycine, and 20% methanol) at 25 V for 12-16 hr. Filters were washed4 times (10 min each) with 10 ml blocking buffer (Tris-buffered salinecontaining 1% non-fat milk and 0.1% TWEEN 20 detergent) and incubatedfor 1 hr with the indicated antibodies (1/1,000 dilution of 8B11 todetect IN, 1/1,000 dilution of JB3904-N to detect RT, and 1/20,000dilution of R2-F to detect Gag) in 10 ml blocking buffer. Membranes werewashed as indicated above and incubated for 1 hr with 1/10,000 dilutionsof alkaline phosphatase-conjugated donkey anti-rabbit (detection of RTand Gag) or anti-mouse (detection of 1N) 1 gG (Pierce; Rockford Ill.) in10 ml blocking buffer. Immunoblots were washed again as indicated,visualized by enhanced chemifluorescence (ECF reagent) development(Amersham) and quantified on a STORM Imaging System with ImageQuantv1.11 (Molecular Dynamics) and Microsoft Excel™ software.

Protein purification and analysis. Recombinant Ty1 RT (Wilhelm et al.,supra, 2000) containing a cleavable N-terminal GST-tag was expressed inE. coli and purified first by affinity chromatography and then by ionexchange chromatography, as described for HIV-1 RT (Le Grice et al.,Meth. Enzymol. 262: 130-44, 1995, which is incorporated herein byreference). The GST-tagged protein was eluted from a GlutathioneSepharose 4B™ gel (Amersham) gravity column with buffer (50 mMHEPES-KOH, pH 7.8, 200 mM KCl, 10% glycerol and 2 mM DTT) containing 30mM reduced glutathione. 50 mM HEPES-KOH, pH 7.0 with 10% glycerol wasadded to the protein solution to lower the pH to 7.1 and the KClconcentration to 25 mM. The recombinant Ty1 RT was further purified overa Resource S™ (Amersham) cation-exchange column using a linear KClgradient (25-500 mM). Recombinant Ty1 RT eluted from the Resource S™column in approximately 250 mM KCl. Purified hetero-dimeric HIV-1 RT (LeGrice et al., supra, 1995) was acquired through the AIDS Research andReference Reagent Program, Division of AIDS, NIAID, NIH. Purified AMV RTwas purchased from Roche Molecular Biochemicals (Mannheim, Germany). TheRT assays performed on the purified RTs were carried out as describedabove, except in the presence of 20 mM KCl, and HIV-1 RT assays wereincubated at 37° C. Each reaction contained 45 ng of purifiedrecombinant Ty1 RT or 30 ng of heterodimeric HIV-1 RT. Under theseconditions, the velocity of the reaction was linear with time and enzymeconcentration.

Results

Isolation and identification of the PMR1 gene. A genetic screen waspreviously developed (Xu and Boeke., supra, 1990) to identify cellularcomponents involved in Ty1 transposition. In one application of thisscreen (Chapman, In Molecular Biology and Genetics (The Johns HopkinsUniversity School of Medicine Press 1991), pages 85 et seq., 1991, whichis incorporated herein by reference; Chapman and Boeke, supra, 1991),yeast strain YH23 (Chapman and Boeke, supra, 1991) containing the URA3Ty1-TRP1 donor plasmid, pX3 (pGal-Ty1 element marked with TRP1; Xu andBoeke, supra, 1987), was mutagenized with ethyl methanesulfate (EMS) andscreened for transposition defects. The transposition assay consists ofa series of replica-plating steps, in which pGal-Ty1 expression andtransposition is induced on galactose containing media, and then scoredby quantifying the number of Trp⁺ plasmid-free colonies. One of the mostsevere and stable transposition-deficient mutants, KM255 (derived by EMSmutagenesis of strain YH23; Chapman, supra, 1991), was characterized.

The KM255 mutation was recessive and was backcrossed, generating sporeclone EBX10A-2B, which was used to isolate the relevant gene bycomplementation cloning. EBX10A-2B, harboring a galactose inducibleTRP1-marked Ty1 element, pX3 was transformed with a LEU2-CEN4-basedgenomic library. Approximately 5,900 of the resulting transformants wereassayed for their transposition phenotype. A single genomic clone, c24-2(FIG. 2), complemented the aberrant transposition phenotype ofEBX10A-2B. Sequence analysis of the clone identified the insert as an11.8 kb fragment of chromosome VII encoding eight predicted open readingframes (ORFs). Subsequent subdivision 8 of the 11.8 kb insert identifieda 4.3 kb fragment, c24-2I, and a smaller 3.1 kb subfragment containingonly the full-length PMR1 gene, c24-2L (see FIG. 2), that fullycomplemented the transposition phenotype. The mutant transpositionphenotype was recapitulated in a strain in which the endogenous PMR1 ORFwas completely replaced by the hygromycin B resistance cassette, hphMX4(Goldstein and McCusker, Yeast 15: 1541-1553, 1999, which isincorporated herein by reference). Supplying PMR1 episomally in clonesc24-2 and c24-2I completely complemented the transposition defect of thepmr1Δ::hphMX4 (pmr1Δ) strain. Furthermore, both haploid parents and alltested diploids that resulted from crossing EBX10A-2B with the pmr1Δstrain were hypersensitive to Mn²⁺, whereas diploids produced bycrossing EBX10A-2B with JB740 (PMR1) were not hypersensitive to Mn²⁺.This result indicates that the original mutation present in EBX10A-2Bresides within PMR1.

Disrupting Mn²⁺ transport of Pmr1p decreases Ty1 transposition. Sincecells lacking PMR1 were found to specifically accumulate Mn²⁺ and Ca²⁺(Lapinskas et al., Mol. Cell. Biol. 15: 1382-1388, 1995, which isincorporated herein by reference), an examination was made as to whetherthe accumulation of either Mn²⁺ or Ca²⁺ was responsible for thetransposition defect. To do this, three Pmr1psingle-amino-acid-substitution mutations were assayed for their abilityto complement the transposition phenotype of the pmr1Δ strain. The Q783Amutation in Pmr1p specifically abolishes Mn²⁺ transport, whereas theD53A mutation prevents Ca²⁺ transport. The D778A mutation abrogates bothtransport activities (Wei et al., J. Biol. Chem. 275: 23927-23932, 2000;Wei et al., Biochemistry 38: 14534-14541, 1999, each of which isincorporated herein by reference). Yeast expressing Pmr1p with eitherthe Q783A or the D778A mutation (both of which abolish Mn²⁺ transport)displayed the mutant transposition phenotype, whereas cells producingPmr1p with the D53A mutation (normal Mn²⁺ transport) showed a wild-typetransposition phenotype. Ty1 transposition was only deficient in thosemutants unable to transport Mn²⁺ into the secretory pathway, indicatingthat the aberrant transposition phenotype is due to cytosolicaccumulation of Mn²⁺.

Synthesis of Ty1 cDNA is decreased in cells lacking PMR1. In order toidentify the point in the Ty1 lifecycle (expression and VLP assembly,reverse transcription, or integration) at which the transposition blockoccurred, the relative steady-state levels of marked-Ty1 RNA in thewild-type and pmr1Δ mutant cells were verified to be equivalent. Toassess whether cDNA was being synthesized in pmr1Δ cells, genomic DNAwas isolated from isogenic strains grown under pGal-Ty1-inducingconditions and relative steady-state levels of Ty1 cDNA were measured.PMR1 cells produced at least 50-fold more Ty1 cDNA than pmr1Δ cells.Moreover, cells expressing mutant forms of Pmr1p unable to transportMn²⁺, D778A and Q783A, produced nearly 20-fold less Ty1 cDNA than cellsable to transport Mn²⁺, wild-type Pmr1p and the D53A mutant. Theseresults indicate that transposition in pmr1Δ mutants is hindered at apoint between translation of the Ty1 proteins and the end of cDNAsynthesis.

VLPs are made in cells lacking PMR1. In order to discern whether thetransposition defect in pmr1Δ cells resulted from altered proteincomponents and/or VLP assembly, VLPs were isolated from PMR1 and pmr1Δcells. Immunoblot analyses and RT assays were performed on fractionsfrom the density gradients on which the VLPs were purified. The amountsof Ty1 RT and Gag present in both sets of VLPs were nearly identical forboth sets of VLPs (see FIG. 3A). While the amounts of integrase infractions 21-22 and 27-28 were slightly less for the VLPs isolated frompmr1Δ cells, the amounts present in the peak fractions for RT activitywere nearly identical to those of the VLPs isolated from the PMR1 cells(FIG. 3A); the small variations observed are typical experimentalvariations seen with this technique. Thus, similar amounts of normallyassembled VLPs are made in PMR1 and pmr1Δ cells. Moreover, the absenceof any high molecular weight species on the immunoblots, together withthe normal ratio of precursor (p49) to processed (p45) Gag, suggestedthat the VLP-associated Ty1 proteins isolated from pmr1Δ cells wereprocessed by the Ty1 protease similarly to those found in VLPs isolatedfrom PMR1 cells.

Mn²⁺ inhibits in vitro RT activity even in excess Mg²⁺. To determinewhether Mn²⁺ could directly alter the activity of Ty1 RT, in vitroreverse transcription assays were performed using VLPs isolated fromPMR1 and pmr1Δ cells. VLPs isolated from pmr1Δ cells incorporatedradiolabeled dGTP into the primer-template (dG)¹²⁻¹⁸-poly(rC) at a ratesimilar to VLPs isolated from wild-type (PMR1) cells (FIG. 3B).Moreover, the RT activity associated with both sets of VLPs wasdecreased to similar extents when Mn²⁺ was substituted for Mg²⁺ as thedivalent cation. Therefore, Ty1 RT produced in pmr1Δ mutant cells wasindistinguishable from RT present in wild-type cells. These resultsindicate that Mn²⁺ directly affects the process of reversetranscription, and possibly the RT itself, since RT is a metal-dependentpolymerase.

Competition experiments between Mn²⁺ and Mg²⁺ (FIG. 4) showed thatexceedingly low levels of Mn²⁺ inhibited the RNA-directed DNA polymeraseactivity of VLPs in the presence of excess Mg²⁺ regardless of whetherthe VLPs were obtained from wild-type or pmr1Δ cells (FIG. 4A). Evenmore dramatic Mn²⁺-dependent inhibition was observed for purifiedhetero-dimeric HUV-1 RT (FIG. 4B), purified AMV RT and purifiedrecombinant Ty1 RT (FIG. 4C). An earlier study hinted that the RT inHIV-1 virions, but not homodimeric HIV-1 RT, was sensitive to Mn²⁺ invitro (Filler and Lever, AIDS Res. Hum. Retroviruses 13: 291-299, 1997,which is incorporated herein by reference). Since RTs are active on bothRNA and DNA templates, and both activities are required for replication,it was important to determine the effect of Mn²⁺ on both templates.Mn²⁺-dependent inhibition was also observed when a DNA template(poly(dC)) was substituted for poly(rC), for both Ty1 VLPs and purifiedrecombinant Ty1 RT, although the extent of inhibition was slightly lessdramatic with the DNA template. Inhibition of Ty1 RT was Mn²⁺-specific;when a control competition experiment was conducted, in which Ca²⁺ wassubstituted for Mn²⁺, no significant inhibition was observed (FIG. 4C).Ratios of Mn²⁺ to Mg²⁺ that reduced RT activity by 50% were calculatedand ranged from as low as 7×10⁻⁴ for HIV-1 RT to 1×10⁻³ for Ty1 RT andto 8×10⁻³ for Ty1 VLPs. Thus, very small quantities of Mn²⁺ had aprofound impact on RT activity in vitro.

An RT mutant insensitive to Mn²⁺-dependent inhibition in vitro. Whilestructural studies led to the proposed two metal model for DNApolymerization, they are speculative and offer little information aboutthe kinetic properties at the two metal-binding sites (sites A and B;see FIG. 1). To discern which of the metal-binding sites contribute mostto the catalytic activity of RT and which site is responsible for theMn²⁺-dependent inhibition, the A site was specifically perturbed. Aprevious study found that Ty1 VLPs containing a mutant form of the RTwith an asparagine replacing an invariant aspartate at position 211(D211N) could carry out reverse transcription in vitro (Uzun andGabriel, supra, 2001). Based on the fact that the mutant residue iscoordinated to metal A, the effect of the D211N mutation is mostly ifnot entirely on the divalent cation at the A site (FIG. 1). Compared towild-type (Wt) Ty1 RT, the D211N Ty1 RT was not inhibited by increasingconcentrations of Mn₂₊ in the presence of excess Mn²⁺ (FIG. 4D). Infact, the modest increase in activity seen when Mn²⁺ is added to D211NRT, which peaks at a ratio of 0.02 (Mn²⁺ concentration to Mg²⁺concentration), likely resulted from saturating the partially occupied Asite (see below). Increased activity at low Mn²⁺ concentrations was alsoobserved for purified M-MuLV RT, with or without its associated RNase H,which incorporated slightly more radiolabeled dGTP into theprimer-template as Mn²⁺ concentrations increased. These results indicatethat Mn²⁺ affected the RT enzyme and not the primer-template or dNTPsubstrates, and further indicate that the B site divalent cation is amajor determinant of RT catalytic activity.

The D211N mutation in Ty1 RT was shown to abolish transposition in vivo(Uzun and Gabriel, supra, 2001). Because the D211N mutant RT wasinsensitive to increasing concentrations of Mn²⁺ in the presence ofexcess Mg²⁺, the transposition of a Ty1 element containing D211N RT wasexamined in pmr1Δ cells. Wild-type and D211N RT Ty1 elements transposedequally poorly in pmr1Δ cells. This was not unexpected because the D211NRT previously was reported to prevent the completion of cDNA synthesisand not the initiation of minus strand strong stop synthesis (Uzun andGabriel, supra, 2001).

Cooperativity of divalent metal ion binding for RTs. To furtherinvestigate the metal-dependent activity of purified recombinant Ty1 RT,kinetic analyses was performed on the wild-type and D211N RTs. At fixedsubstrate concentrations, Mg²⁺ activated the wild-type (FIG. 5A) and theD211N (FIG. 5B) RTs, and for both enzymes the Mg²⁺-dependent activationcurves were sigmoidal. A similar sigmoidal activation curve was observedfor purified hetero-dimeric HUV-1 RT. Moreover, Mn²⁺ activated thewild-type (FIG. 5C) and D211N (FIG. 5D) Ty1 RTs. For both enzymes, theMn²⁺-dependent activation curves were also sigmoidal. From inspection ofthe four activation curves, it was evident that much more Mg²⁺ wasneeded to achieve half maximal activity for the D211N RT compared to thewild-type RT. Conversely, much less Mn²⁺ is required to reach peakactivity for the D211N RT relative to the wild-type RT. The sigmoidalactivation curves precluded direct determination of Michaelis constantsfor Mg²⁺ and Mn²⁺.

However, a fit of the data to the Hill equation yielded the average Hillcoefficient (n), the macroscopic dissociation constants (K_(0.5)) andthe maximum velocity or catalytic turnover (k_(cat)) for each divalentcation with each RT (summarized in Table 1). While the wild-type andD211N Ty1 RTs support similar maximal velocities for their preferredmetal ion, they have very different affinities for that same metal ion.The wild-type RT has 50-fold higher affinity for Mg²⁺ than does D211NRT. In contrast, D211N RT has 1000-fold higher affinity for Mn²⁺ thanwild-type RT. The Hill plot (FIG. 6E) shows the fit of the aboveactivation curves to the Hill equation. Linear regression analysis ofeach sigmoidal activation curve yielded Hill coefficients ranging from1.7-2.3, indicating that there are at least two metal-binding sites andthat there is positive cooperativity for metal-binding.

Ty1 retrotransposons, like retroviruses, exist as genome parasiteswithin a host cell, and as such, their lifecycle can be influenceddramatically by the host cell environment. They encode an RT thatenables transposition by converting the Ty1 transcript into afull-length cDNA copy. RTs and many aspects of the reverse transcriptionmechanism, mainly base-pairing interactions, require the presence ofspecific divalent cations, such as Mg²⁺ or Mn²⁺. Cells lacking PMR1 weredeficient for Ty1 transposition. Moreover, the transposition phenotypedepends on the ability of Pmr1p to transport Mn²⁺, and not Ca²⁺. Thus,it is unlikely that Pmr1p functions directly in the Ty1 lifecycle.Rather, Pmr1p regulates cytosolic Mn²⁺ homeostasis, which subsequentlyinfluences one or more aspects of transposition.

Polymerases and specifically RTs can carry out their enzymatic functionsin the presence of Mg²⁺ or Mn²⁺. However, the use of Mn²⁺ instead ofMg²⁺ as a divalent cation tends to decrease the enzymatic activity andthe fidelity of these enzymes. Most RTs studied exhibit a preference forMg²⁺ over Mn²⁺. The enzymatic activity of purified recombinant Ty1 RTwith Mg²⁺ was reported to be 3 fold that with Mn²⁺ (Wilhelm et al.,supra, 2000). Under saturating conditions of individual metal ions,similar differences in activity with the VLP preparations and purifiedrecombinant Ty1 RTs were observed (FIG. 5; see, also, Table 1, below).However, under more biologically relevant conditions (free divalentcation concentrations less than 6 mM) for wild type Ty1 RT,Mg²⁺-dependent activation was nearly 20-fold higher than that ofMn²⁺-dependent activation (compare FIGS. 5A and 5C).

Ty1 cDNA production was markedly reduced in pmr1Δ cells, while VLPassembly remained unaffected. Moreover, pmr1Δ mutants specificallyimpaired in Mn²⁺ transport demonstrated a similar defect in cDNAproduction. These genetic findings indicate that the transpositiondefect in pmr1Δ mutants is due to accumulation of cytoplasmic Mn²⁺ andsubsequent inhibition of reverse transcription of Ty1 RNA into cDNA.Also provided is the first direct kinetic evidence for a dual divalentcation requirement at the active site of a polymerase, specifically anRT; there are at least two metal-binding sites, and positivecooperativity exists between these sites.

Four possible explanations for the Ty1 transposition phenotype of pmr1Δmutants were considered. 1) alternative activation (homo-ionicactivation/hetero-ionic inhibition) based on binding of two metal ionsat sites with differential affinitial and effects on catalytic activity(see Table 2, below); 2) the Mn²⁺ accumulation could interfere withprimer-template interactions required by the RT to generate afull-length cDNA; 3) the accumulation of Mn²⁺ directly alters thefidelity of the Ty1 RT to such an extent that cDNA synthesis isinhibited; 4) the accumulation of Mn²⁺ could inhibit the activity of theRNase H domain of the RT. The first explanation is favored because ofthe striking parallels between the crystal structure and sequencecomparisons (discussed above) and the biochemical finding that Mn²⁺, butnot Ca²⁺, directly inhibited the RT activity of Ty1 RT in vitro even inthe presence of excess Mg²⁺. Second, in vivo estimates of divalent metalions for yeast were considered; the total intracellular concentrationsof Mn²⁺ were estimated to be 50 μM in PMR1 cells and 250 μM in pmr1Δcells (calculated from Lapinskas et al., supra, 1995), while theintracellular concentration of free Mn²⁺ was much less, in the lowmicromolar range (Ash and Schramm., J. Biol. Chem. 257, 9261-9264, 1982;Mandal et al., J. Biol. Chem. 275, 23933-23938, 2000). The concentrationof free Mg²⁺ in cells is estimated at 0.1-1 mM (calculated from Beeleret al., Biochim. Biophys. Acta 1323: 310-318, 1997), while the totalMg²⁺ concentration in yeast cells may be as great as 33 mM (calculatedfrom Graschopf et al., J. Biol. Chem. 276: 16216-16222, 2001). The ratioof the estimated free intracellular Mn²⁺ to Mn²⁺ concentrations in yeast(1 μM/1 mM=0.001) falls on the steepest segment of the Mn²⁺ inhibitioncurve (FIG. 4). However, when an attempt is made to estimate the freeMn²⁺ and Mg²⁺ ion concentrations in wild-type and pmr1Δ yeast cells, adecrease of only 2-4 fold in activity is predicted. A more drastic(20-40 fold) decrease in cDNA synthesis was observed. However,successful completion of cDNA synthesis requires synthesis of 12 kb ofDNA and several complex strand transfers and priming events. Therefore,a modest decrease in RT activity could result in a more profound overallcDNA synthesis defect. Third, kinetic studies on the wild-type and D211NTy1 RTs indicate that at least two highly cooperative metal-bindingsites are present in each enzyme (FIG. 5E). Fourth, the D211N mutationaffects the affinity for each type of metal ion, but not the overallV_(Max) of the reaction or K_(cat) relative to wild-type RT (Table 1,below). Finally, Mn²⁺ inhibition was observed with purified Ty1, AMV andHIV-1 RT, but not with M-MuLV RT. This result indicates that Mn²⁺inhibition can be utilized to selectively alter the activity of a subsetof all, RTs, and suggests that other divalent cations also may be usefulfor altering RT activity. Thus, the effect appears to be on the proteinand not on the primer-template, arguing against the second explanation.Additionally, the interactions necessary for substrate annealing werelikely not affected by the trace amounts of Mn²⁺ with such a largeexcess of Mg²⁺ present, especially since M-MuLV RT incorporationremained unaffected. While the homopolymer-based RT assay cannot ruleout the possibility that Mn²⁺ has additional effects on RT fidelityand/or RNase H activity, for example, by disrupting priming events thatoccur during strand transfer, the 10-fold reduction in incorporationrate in a homopolymer assay with a single dNTP cannot be explainedsolely by such an explanation. Preliminary examination of the fidelityof Ty1 RT in wild-type and pmr1Δ mutant cells as well as the affects ofMn²⁺ accumulation on RNase H activity indicates that the accumulation ofMn²⁺ in the pmr1Δ cells alters the in vivo fidelity of the Ty1 RT andalso can effect Ty1 RNase H activity.

All possible combinations of high and low affinities for the two metalions at the A and B sites of RT were modeled, and a single scenariocompatible with all of the kinetic data was obtained (Table 2, below).Under conditions where little or no Mn²⁺ was present (Mn²⁺ concentration<<K_(0.5)), Mg²⁺ ions occupied both the A and B sites within the RTactive site, resulting in a high level of activation (FIGS. 5A and 5B).Similarly, when excess Mr²⁺ was present (Mn²⁺ concentration >>K_(0.5)),Mn²⁺ ions occupied both A and B sites, resulting in a low level ofactivation (FIGS. 5C and 5D). However, when a small amount of Mn waspresent (Mn²⁺ concentration≈K_(0.5)) for the wild-type RT, the A sitewas likely occupied by a Mg²⁺ ion, and the B site likely contained atightly-bound Mn²⁺ ion, resulting in a low level of activation orinhibition (Table 2). On the other hand, when a small amount of Mn²⁺ waspresent (Mn²⁺ concentration≈K_(0.5)) for the D211N RT, the A site waslikely occupied by a Mn²⁺ ion, and the B site likely contained a Mg²⁺ion, which resulted in a high level of activation. These predictions arebased on the fact that increasing amounts of Mn²⁺ inhibited thewild-type RT (FIG. 4), but not the D211N RT (FIG. 4D). The D211Nmutation appeared to dramatically bias the divalent cation affinity atthe A site toward Mn²⁺, allowing Mg²⁺ to saturate the B site. The peakin activity at intermediate levels of Mn²⁺ (FIG. 4D) was likely due tothe saturation of the A site with Mn²⁺, and the presence of Mg²⁺ at theB site. Based on the present data, it is predicted that the higheraffinity site for Mn²⁺ (the B site) in the wild-type RT has the greatestimpact on the V_(Max) of the enzymatic reaction. When Mg²⁺ is bound, ahigh V_(Max) is obtained, and when Mn²⁺ is bound, a lower V_(Max) canoccur because the smaller Mg²⁺ ion at the B site interacts morefavorably than a Mn²⁺ ion sterically with the pyrophosphate leavinggroup and the enzyme. To explain the reversal of metal ion affinitiesthat occurred by replacing the aspartate at position 211 with asparagine(FIG. 1), it is likely that the D211N mutation provides a nitrogenligand to the A site metal, which Mn²⁺ is much more likely than Mg²⁺ tocoordinate (Bock et al., J. Amer. Chem. Soc. 121, 7360-7372, 1999).Tighter Mn²⁺ binding can result from avoiding the clustering of too manyanionic ligands near the metal.

In summary, Pmr1p was identified as an indirect host factor involved inTy1 retrotransposition. Mutations in PMR1 that specifically abolishedMn²⁺ (but not Ca²⁺) transport decreased the frequency of Ty1transposition to the same extent as a complete deletion of PMR1.Moreover, deletion of PMR1, or simply the lack of Mn²⁺ transport,dramatically decreased the relative amount of Ty1 cDNA produced in vivo.In addition, low levels of Mn²⁺ relative to Mn²⁺ dramatically inhibitedRT activity associated with Ty1 VLPs, as well as purified recombinantTy1 RT, AMV RT and HIV-1 RT in vitro. Since Pmr1p regulates cytosolicMn²⁺ homeostasis, the present results indicate that accumulation of Mn²⁺in pmr1D cells inhibits the steps required to reverse transcribe the Ty1RNA into the cDNA copy of the element, thus providing a previouslyundescribed target for affecting RT activity and, therefore, retroviralreplication in cells by perturbing cytoplasmic metal ion concentrationsin a target cell. These present results also reinforce the importance ofmetal ion clusters in catalysis, and support the concept that targetingsuch clusters can be useful for identifying viral replication inhibitors(Filler and Lever, supra, 1997).

EXAMPLE 2 High Throughput Screening Assay for Agents that AlterManganese Trasporter Activity

This example provides a high throughput assay using yeast cellstransformed to express a human Prm1 divalent cation transportingprotein.

As disclosed in Example 1, the yeast retrovirus-like element Ty1 cannotreplicate in pmr1 mutant of yeast due to defective pumping of manganeseion by the mutant Pmr1 transporter. Elevated Mn²⁺ concentration in themutant cells interferes with reverse transcription through combinedeffects on the RT and RNAse H activities, and in vitro studiesdemonstrated that HIV-1 RT, like Ty1 RT, also is exquisitely sensitiveto inhibition by manganese ion (Example 1); Pmr1p is highly conservedfrom yeast to man.

This Example provides a drug screening assay using living yeast cells tofacilitate identification of agents that alter manganese ion transportof a human Pmrp transporter, thus providing a drug that can mimic theeffect of the mutant Pmrp1 transporter and can be useful as to inhibitretrotransposable element replication, for example, HIV-1 replication,wherein the agent can be useful for immune restoration in AIDS patients.Using the disclosed high throughput assay, agents that interfere withthe manganese transporting function of the human Pmr1 transporterprotein can be identified. Such agents that can be examined in vitro toconfirm they affect the human (or yeast) Pmr1p in vitro, then can befurther examined for the ability to inhibit HIV multiplication in asingle round infection assay (Roos et al., Virology 273: 307-315, 2000,which is incorporated herein by reference) or using a p24-basedmultiplication assay.

The exemplified assay, which utilizes a 96 well based format to examineyeast cell growth in the presence or absence of test agents, is based onthe requirement of an sod1 mutant yeast cell of lysine and methioninefor growth in aerobic medium, whereas an sod1 pmr1 double mutant doesnot have such requirements. There are enzymes in the lysine andmethionine biosynthetic pathways that are super-sensitive to superoxideanion and associated free radicals (Lapinskas et al., supra, 1995). Thebasis of the present assay is that elevated redox-active Mn²⁺ ion in thesod1 pmr1 double mutant can act as a “chemical” SOD activity in pmr1mutants such that the pmr1 mutation suppresses the damage caused bysuperoxides and related free radicals.

Compound screen. A yeast strain was constructed having a genotypeincluding sod1::kanAMpmr1::hygAMX uar3. This strain was separatelytransformed with two URA3 centromeric yeast vectors, one expressinghuman Pmr1 (EBY 115c) and the other an empty vector (EBY116B), whichserves as a positive control for growth. Test agents that allow thehPmr1p strain grow as well as this positive control strain are selectedas drug candidates useful for inhibiting RT activity and retroviralmultiplication.

A Hydra®-96 FlexChem® Microdispenser liquid handling robot (ApogentDiscoveries; Hudson N.H.) to assemble 96 well plates containing 0.1 mlaliquots of medium seeded with approximately 100 cells of theappropriate genotype. The medium is a minimal medium lacking lysine,methionine and uracil, the latter to select the hPmr1p plasmid. Themedium also can contain hygromycin and carbenicillin, if necessary, toprevent contaminants from growing. Four ul aliquots of test agent(approximately 400 uM in DMSO) from the PRIME-Collection 2000™combinatorial library (Chembridge Corp.; San Diego Calif.) or selectedfrom the ChemBridge™ Master Database, for example, are added to a well;duplicates, triplicates, and/or examination of a test agent at two ormore concentrations also can be included. Typical hit rates fortransporters in assays similar to the present assay range from about 1in 1000 to 1 in 10,000. The use of the Hydra®-96 FlexChem®Microdispenser liquid handling robot to aliquot the test agent, mix thetest agent with the culture, dilute the drug, etc., minimizes the amountof labor and the likelihood of pipetting errors. Using the exemplifiedassay, a team of two people can process several thousand compounds perweek.

Under the growth conditions, there is a 48 to 72 hr delay between thetime when the control strain (the one without hPmr1p) grows to a maximaloptical density and when the experimental strain grows. This period oftimes provides an excellent dynamic range such that, even if a drug thatis only, for example, about 10% effective, the assay can allowidentification of the activity. The readout for growth is performedusing a 96 well spectrophotometer, can incorporate a computerized outputwith an algorithm for subtracting background. For example, each 96 wellplate can contain 80 test agents, one in each well of columns 2 to 11,and solvent alone in columns 1 and 12, such that the wells of columns 1and 12 can be averaged and the mean background can be automaticallysubtracted from each well. All of the growth data can be monitoredseveral times over the 48 to 72 hr period, thus allowing a determinationof the growth kinetics.

Primary positives that are identified can be further examined in abattery of secondary assays. First, the growth test can be repeatedusing a broader range of test agent concentrations. Second, positivescan be further tested against a similar strain to that described above,but containing a wild-type SOD1 gene. Loss of PMR1 function makes cellssupersensitive to growth in high Mn⁺² ion, thus providing a secondsimple growth test that can be performed in the presence of various drugconcentrations. If desired, additional yeast strains can be madecontaining the yeast Pmr1 (or other Pmr1 transporter) instead of thehuman Pmr1p. Positive test agents also can be examined for the abilityto interfere with Ty1 retrotransposition in strains expressing yeast orhuman Pmr1 protein.

A number of negative controls can be performed, either in separateassays or in parallel with any particular assay, including, for example,addition of the vehicle in which the compounds are suspended (DMSO)added alone to the wells. Positive controls include supplementation ofthe medium with uracil, which allows loss of the PMR1 plasmid andluxuriant growth (as would be expected for a positive test agent). Noinhibitors of hPMR1 or yeast PMR1 have not previously been identifiedand, therefore, such a positive control compound that directly inhibitsthe transporter is not available.

In vitro testing of identified agents. Positive agents identified usingthe above described screening assay can be further examined in vitro forthe ability to alter manganese ion transport. As assays similar to thatused to detect the transport of Ca²⁺ into vesicles isolated from yeastcells expressing either yeast or human Pmr1 protein (Sorin et al., J.Biol Chem. 272: 9895-901, 1997; Ton et al., J. Biol. Chem. 277:6422-6427, 2002, each of which incorporated herein by reference) can beused to measure manganese transport. The described assay is based onimport of ⁴⁵Ca²⁺ into vesicle isolated from the yeast cells. This invitro assay can be used to examine whether the effect of any agentidentified in the screening assay is a direct effect on the Pmr1p, orcan be used to determine whether the agent is active specificallyagainst the human Pmr1p transporter, or whether in more generallyeffects, for example, the yeast and human Pmr1p transporters.

Testing compounds for anti-HIV-1 activity. A simple single-roundinfection assay based on the activation of an SIV LTR-luciferasereporter gene can be used to determine whether an identified agent caninhibit HIV-1 RT activity (Roos et al., supra, 2000). As the drug ispredicted to affect the reverse transcription step, it would be expectedto be active only in the target cell and not in the producer cells. Theluciferase assay can easily be carried out in a high throughput format.Further, any positives identified in the single-round infection assaycan be examined using a multi-round assay, for example, a p24 assay (orRT assay) following a low multiplicity of infection (MOI). Methods ofperforming p24 assays and RT assays are well known and routine in theart (see, for example, Goff et al., J. Virol. 38: 239-248, 1981, whichis incorporated herein by reference; describing an RT assay), and can beperformed, for example, using commercially available kits (e.g., anAlliance® HIV p24 RIA kit; Perkin Elmer; prod. no. NEK040001KT).

Although the invention has been described with reference to the aboveexample, it will be understood that modifications and variations areencompassed within the spirit and scope of the invention. Accordingly,the invention is limited only by the claims, which follow Tables 1 and2.

TABLE 1 Kinetic Constants of Metal Activation of Wt. and D211N Ty1Reverse transcriptases K_(0.5)Mg²⁺ K_(0.5)Mn²⁺ k_(cat)Mg²⁺ k_(cat)Mn²⁺Ty1 RT _(n)Mg2+ _(n)Mn2+ (mM) (mM) (hr⁻¹) (hr⁻¹) Wt. 1.7 ± 0.1 2.3 ± 0.20.13 ± 0.04 19.9 ± 1.6  1.61 ± 0.32 0.56 ± 0.07 D211N 2.1 ± 0.1 2.1 ±0.1 6.3 ± 0.2 0.02 ± 0.003 1.60 ± 0.28 0.57 ± 0.03

TABLE 2 Proposed divalent cation binding affinities and activities forRTs A site B site Wt. Ty1 RT very low affinity for Mn²⁺ high affinityfor Mn²⁺ very high affinity for Mg²⁺ low affinity for Mg²⁺ D211N Ty1 RTvery high affinity for Mn²⁺ high affinity for Mn²⁺ very low affinity forMg²⁺ low affinity for Mg²⁺ metal activation of Ty1 RT A site + B site =activity comment Mg²⁺ Mg²⁺ high Mg²⁺ Mn²⁺ low unlikely for D211N Mn²⁺Mg²⁺ high unlikely for Wt. Mn²⁺ Mn²⁺ low

1. A method of identifying an agent that reduces the activity of reversetranscriptase comprising: a) contacting a cell comprising a divalentcation transporting protein with a test agent; b) detecting a change inthe concentration of intracellular manganese ions in the cell aftercontact with the test agent as compared to the concentration ofmanganese ion levels in the absence of the test agent; and c) detectingreverse transcriptase activity in the cell using a polyribonucleotide orpolydeoxyribonucleotide template for reverse transcriptase activity,wherein an increase in the concentration of intracellular manganese ionsin the cell after contact with the test agent, and a decrease inactivity of reverse transcriptase is indicative of an agent that reducesthe activity of reverse transcriptase.
 2. The method of claim 1, whereinthe cell comprises an isolated cell membrane.
 3. The method of claim 2,wherein the cell membrane comprises a eukaryotic cell membrane.
 4. Themethod of claim 3, wherein the eukaryotic cell membrane comprises ayeast cell membrane or a mammalian cell membrane.
 5. The method of claim3, wherein the eukaryotic cell membrane comprises a human cell membrane.6. The method of claim 1, wherein contacting the cell comprisescontacting a cell comprising a cell membrane.
 7. The method of claim 6,wherein the cell comprises a yeast cell.
 8. The method of claim 6,wherein the cell comprises a human cell.
 9. The method of 6, wherein thecell comprises a T lymphocyte.
 10. The method of claim 1, wherein thecell comprises a divalent cation transport protein comprising an ATPase.11. The method of claim 1, wherein the cell comprises a divalent cationtransport protein comprising a P-type ATPase.
 12. The method of claim11, wherein the ATPase is a Pmr1p protein or a homolog thereof.
 13. Themethod of claim 1, wherein the test agent does not alter transport of adivalent cation other than manganese ions by a divalent cationtransporting protein.
 14. The method of claim 1, wherein the agentreduces or inhibits manganese ion transport out of a cell.
 15. Themethod of claim 1, wherein the polyribonucleotide orpolydeoxyribonucleotide template comprises a nucleotide sequence of aretrotransposable element, and wherein further the retrotransposableelement is a human immunodeficiency virus (HIV).
 16. The method of claim1, wherein the polyribonucleotide or polydeoxyribonucleotide templatecomprises a nucleotide sequence of a retrotransposable element, andwherein further the retrotransposable element is a Ty retrotransposon.